Agonistic binding molecules to the human OX40 receptor

ABSTRACT

The present invention provides binding molecules, such as human binding molecules, that bind to and stimulate the human OX40-receptor. The invention also provides nucleic acids encoding such binding molecules. Methods for producing such binding molecules are also provided by the present invention. The binding molecules and nucleic acids are useful in the stimulation of human T-cells and can be used to enhance antigen-specific immune responses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 10/517,941, filed Dec. 13, 2004, which is a national phaseentry under 35 U.S.C. §371 of International Patent Application No.PCT/EP03/06341, filed Jun. 13, 2003, published in English asInternational Patent Publication No. WO 03/106498 on Dec. 24, 2003,which claims the benefit under of International Patent Appl'n No.PCT/NL02/00389 filed Jun. 13, 2002, the contents of the entirety of eachof which is incorporated herein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5)—SEQUENCE LISTING SUBMITTEDON COMPACT DISC

Pursuant to 37 C.F.R. §1.52(e)(1)(ii), a compact disc containing anelectronic version of the Sequence Listing has been submittedconcomitant with this application, the contents of which are herebyincorporated by reference. A second compact disc is submitted and is anidentical copy of the first compact disc. The discs are labelled “copy1” and “copy 2,” respectively, and each disc contains one file entitled“0077 (OX40) Nov. 11, 2005 version.ST25.txt” which is 90 KB and createdon Nov. 16, 2005.

FIELD OF THE INVENTION

The present invention relates generally to the field of biotechnologyand more specifically to the field of medicine, more in particular, toagonistic binding molecules capable of specifically binding to the humanOX40-receptor. The binding molecules are useful in immunotherapy.

BACKGROUND

The OX40-receptor (OX4OR) (also known as CD134, ACT-4, ACT35) is amember of the TNF receptor family which is expressed on activated CD4+T-cells (see WO 95/12673). Triggering of this receptor via theOX40-ligand, named OX40L, gp34 or ACT-4-ligand, present on activatedB-cells and dendritic cells, enhances the proliferation of CD4+ T-cellsduring an immune response and influences the formation of CD4+ memoryT-cells. Furthermore, the OX40R-OX40L system mediates adhesion ofactivated T-cells to endothelial cells, thus directing the activatedCD4+ T-cells to the site of inflammation.

Inflammatory and autoimmune diseases, such as rheumatoid arthritis andinflammatory bowel disease, are characterized by an infiltration ofactivated T-cells at the site of inflammation, which is believed toorchestrate the response leading to chronic tissue destruction. Inpatients with inflammatory bowel disease, OX40+ CD4+ T-cells can befound in the gut associated with sites of inflammation. In addition, inpatients suffering from acute graft-vs-host-disease, elevated levels ofOX40+ peripheral CD4+ T-cells are present in peripheral blood. Inrheumatoid arthritis patients, OX40+ CD4+ T-cells are present insynovial fluid, while they are virtually absent from peripheral blood.Furthermore, OX40+ CD4+ T-cells are found in inflamed synovial tissue inaddition to cells expressing the ligand for the OX40-receptor. This isin contrast to patients suffering from osteoarthritis, a joint diseasethat is not mediated by inflammation, where both cell types could not befound in significant numbers.

Thus, in patients suffering from several inflammatory disorders elevatedlevels of OX40+ CD4+ T-cells are present at sites of inflammation,indicating that these cells may be involved in progression of autoimmunedisease. A blockade of the OX40R-OX40L pathway using antibodies orfusion proteins has led to the attenuation of disease progression inseveral animal models of autoimmune disease.

Besides their presence in autoimmune diseases, it has been shown thatOX40+ T-cells are present within tumor lesions containing tumorinfiltrating lymphocytes and in tumor cell positive draining lymph nodes(Weinberg et al., 2000). It was shown in several tumor models in micethat engagement of the OX40-receptor in vivo during tumor primingsignificantly delayed and prevented the appearance of tumors as comparedto control treated mice (Weinberg et al., 2000). Hence, it has beencontemplated to enhance the immune response of a mammal to an antigen byengaging the OX40-receptor by administering an OX40-receptor bindingagent (WO 99/42585; Weinberg et al., 2000). One possibility is to use anatural ligand of the OX40-receptor, i.e. the OX40-ligand, or fusionproteins thereof as an OX40-receptor binding ligand. Such proteinshowever have a fixed affinity for the receptor that is not easilychanged, may not have the circulatory retention time to exert thedesired therapeutic effect, and may give rise to immunogenicity(Weinberg et al., 2000).

Another possibility to stimulate T-cells by virtue of the OX40-receptorpathway, is to use antibodies against this receptor (Kaleeba et al.,1998; Weinberg et al., 2000). A rat anti-mouse OX40-receptor antibodynamed OX86 (Al-Shamkhani et al., 1996) appeared to engage theOX40-receptor in murine tumor models (Weinberg et al., 2000; U.S. Pat.No. 6,312,700).

To our knowledge agonistic antibodies, particularly human agonisticantibodies, that are capable of stimulating the human OX40-receptor havenot been disclosed in the art. Furthermore, it is well known thatnon-human antibodies are limited in their use in vivo in humans.Problems associated with administration of non-human antibodies tohumans are inter alia short serum half life, an inability to triggercertain human effector functions and elicitation of an unwanted dramaticimmune response against the non-human antibody in a human.

In general, attempts to overcome the problems associated with use offully non-human antibodies in humans, have involved geneticallyengineering the antibodies to be more “human-like.” A first stage in thehumanization process was preparing chimeric antibodies, i.e. antibodiesin which the variable regions of the antibody chains are derived fromthe non-human species and the constant regions of the antibody chainsare human-derived. Subsequently, domains between the variable domainswhich specify the antigen binding were replaced by their humancounterparts leading to so-called humanized antibodies. A disadvantageof these chimeric and humanized antibodies is that they still retainsome non-human sequences and therefore still elicit an unwanted immunereaction, especially when administered for prolonged periods.

In the light of the above, there is still a need for human antibodiesthat stimulate the human OX40-receptor. These antibodies can be usefulin inter alia the treatment and/or prevention of tumours in humans.

SUMMARY OF THE INVENTION

The invention provides agonistic binding molecules capable ofspecifically binding to the human OX40-receptor. In a preferredembodiment, said binding molecules are human binding molecules.Furthermore, the invention pertains to nucleic acid molecules encodingat least the binding region of the binding molecules. The inventionfurther provides for the use of the binding molecules or nucleic acidsfor enhancing the immune response in a human, for use in the treatmentof the human or animal body, and for the preparation of a medicament totreat a human having or at risk of developing a disorder or disease suchas a neoplastic disorder or disease.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the binding of anti-human OX40-receptor phage antibodies,that were selected using immobilized human OX40-Ig fusion protein, tohuman OX40-Ig fusion protein coated to ELISA plates. The Y-axis showsthe absorbance at 492 nm.

FIG. 2 shows the binding of anti-human OX40-receptor phage antibodies,that were selected using immobilized human OX40-Ig fusion protein, tohuman OX40-receptor transfected PER.C6™ cells. In each picture the groupof cells on the left are control transfected PER.C6™ cells and the groupof cells on the right are OX40-receptor transfected PER.C6™ cells. Theupper left picture shows binding of a control phage antibody directedagainst thyroglobulin.

In FIG. 3 the binding of anti-human OX40-receptor phage antibodies,selected using immobilized human OX40-Ig fusion protein, to OX40+ CD4+T-cells is shown. In FIG. 3A the binding of the selected phageantibodies to a subset of CD4+ T-cells within tonsil mononuclear cellsis shown. In FIG. 3B the binding of the phage antibodies to a subset ofCD4+ T-cells within synovial fluid mononuclear cells is shown. In FIG.3C the binding of the selected phage antibodies to peripheral blood CD4+T-cells is displayed. The upper FACS plot in FIGS. 3A, 3B and 3C shows acontrol staining on CD4+ T cells using a PE-labelled mouse anti-humanOX40 antibody.

FIG. 4 shows the binding of anti-human OX40-receptor phage antibodies,selected using OX40+ CD4+ T-cells, to OX40+ CD4+ T-cells (see FIG. 4A)and to human OX40-receptor transfected PER.C6™ cells (see FIG. 4B).

FIG. 5 shows the nucleotide sequence (SEQ ID NO:1) and amino acidsequence (SEQ ID NO:2) of the scFv called SC02008. The heavy chain CDR3region is underlined.

FIG. 6 shows the nucleotide sequence (SEQ ID NO:3) and amino acidsequence (SEQ ID NO:4) of the scFv called SC02009. The heavy chain CDR3region is underlined.

FIG. 7 shows the nucleotide sequence (SEQ ID NO:5) and amino acidsequence (SEQ ID NO:6) of the scFv called SC02010. The heavy chain CDR3region is underlined.

FIG. 8 shows the nucleotide sequence (SEQ ID NO:7) and amino acidsequence (SEQ ID NO:8) of the scFv called SC02011. The heavy chain CDR3region is underlined.

FIG. 9 shows the nucleotide sequence (SEQ ID NO:9) and amino acidsequence (SEQ ID NO:10) of the scFv called SC02012. The heavy chain CDR3region is underlined.

FIG. 10 shows the nucleotide sequence (SEQ ID NO:11) and amino acidsequence (SEQ ID NO:12) of the scFv called SC02021. The heavy chain CDR3region is underlined.

FIG. 11 shows the nucleotide sequence (SEQ ID NO:13) and amino acidsequence (SEQ ID NO:14) of the scFv called SC02022. The heavy chain CDR3region is underlined.

FIG. 12 shows the nucleotide sequence (SEQ ID NO:15) and amino acidsequence (SEQ ID NO:16) of the scFv called SC02023. The heavy chain CDR3region is underlined.

FIG. 13 shows the construction of the bivalent scFv expression vectorpPICZbiFVH. In FIG. 13A the vector pPICZαB is shown and in FIG. 13B thebivalent scFv expression vector pPicZbiFVH is shown. FIG. 13C shows thecloning strategy of scFv's into pPicZbiFVH.

FIG. 14 shows the functional activity of the anti-human OX40-receptorbivalent scFv's SC02008 and SC02023 in an in vitro T-cell costimulationassay. FIG. 14A shows the stimulation assay for the bivalent scFvSC02008 and FIG. 14B shows the stimulation assay for the bivalent scFvSC02023.

FIG. 15 shows the binding of human IgG molecules called 008, 011, 021and 023 to human OX40-receptor transfected PER.C6™ cells.

DETAILED DESCRIPTION OF THE INVENTION

Herebelow follow definitions of terms as used in the invention

Definitions Agonistic Binding Molecule

The term “agonistic binding molecule” as used herein in general refersto a binding molecule which, when combined with a receptor, e.g. theOX40-receptor, on a cell, is capable of binding to the receptor and iscapable of initiating/mimicking/stimulating a reaction or activity thatis similar to or the same as that initiated/mimicked/stimulated by thereceptor's natural ligand, e.g. the OX40-ligand. An agonistic bindingmolecule of the OX40-receptor is capable of immunospecifically bindingto the OX40-receptor expressed by activated CD4+ T-cells, and is capableof inducing/augmenting/enhancing/stimulating the activation of a signaltransduction pathway associated with the OX40-receptor such as forinstance the activation of the activated CD4+ T-cells.

Agonistic binding molecules are capable ofinducing/augmenting/enhancing/stimulating any or all of, but not limitedto, the following responses: proliferation of CD4+ T-cells during animmune response, stimulation of cytokine production, proliferation ofTh1 or Th2 effector cells, development of a Th2 response, generation ofCD4+ memory T cells. An agonistic binding molecule mayinduce/enhance/stimulate/augment any one or more of the responses by 5%,10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, morepreferably 70%, 80%, 85%, and most preferably 90%, 95%, 99%, or 100%. hiparticular, an agonistic binding molecule that is capable ofinducing/enhancing/stimulating/augmenting an activated CD4+ T-cellactivates an activated CD4+ T-cell 1-5 fold, 5-10 fold, 10-20 fold, ormore than 20 fold as compared to the ability of the agonistic bindingmolecule to activate a resting T-cell, i.e. T-cells which do not expressor express low to undetectable levels of the T-cell activation markerCD4. Methods for determining theactivation/stimulation/induction/enhancement are known in the art andinclude, but are not limited to, antigen specific proliferation assays,cytokine ELISA assays, elispot assays, detection of antigen specificT-cells using flow cytometry methods employing Major HistocompatibilityComplex (MHC) peptide tetramers. The agonistic binding molecules arepreferably against epitopes within the extracellular domain of theOX40-receptor. The term “agonistic binding molecule” as used hereincovers inter alia agonistic human anti-OX40-receptor monoclonalantibodies or parts thereof and agonistic human anti-OX40-receptorcompositions with polyepitopic specificity.

Amino Acid Sequence

The term “amino acid sequence” as used herein refers to naturallyoccurring or synthetic molecules and to a peptide, oligopeptide,polypeptide or protein sequence.

Binding Molecule

As used herein the term “binding molecule” refers to an intactimmunoglobulin including monoclonal antibodies, such as chimeric,humanized or human monoclonal antibodies, or to an antigen-bindingand/or variable domain comprising fragment of an immunoglobulin thatcompetes with the intact immunoglobulin for specific binding to thebinding partner of the immunoglobulin, e.g. OX40-receptor. Regardless ofstructure, the antigen-binding fragment binds with the same antigen thatis recognized by the intact immunoglobulin. An antigen-binding fragmentcan comprise a peptide or polypeptide comprising an amino acid sequenceof at least 2 contiguous amino acid residues, at least 5 contiguousamino acid residues, at least 10 contiguous amino acid residues, atleast 15 contiguous amino acid residues, at least 20 contiguous aminoacid residues, at least 25 contiguous amino acid residues, at least 30contiguous amino acid residues, at least 35 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least contiguous 80 aminoacid residues, at least contiguous 90 amino acid residues, at leastcontiguous 100 amino acid residues, at least contiguous 125 amino acidresidues, at least 150 contiguous amino acid residues, at leastcontiguous 175 amino acid residues, at least 200 contiguous amino acidresidues, or at least contiguous 250 amino acid residues of the aminoacid sequence of the binding molecule.

The term “binding molecule,” as used herein also includes theimmunoglobulin classes and subclasses known in the art. Depending on theamino acid sequence of the constant domain of their heavy chains,binding molecules can be divided into the five major classes of intactantibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgA1, IgA2, IgG1,IgG2, IgG3 and IgG4.

Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)2, Fv,dAb, Fd, complementarity determining region (CDR) fragments,single-chain antibodies (scFv), bivalent single-chain antibodies,diabodies, triabodies, tetrabodies, (poly)peptides that contain at leasta fragment of an immunoglobulin that is sufficient to confer specificantigen binding to the (poly)peptide, etc. The above fragments may beproduced synthetically or by enzymatic or chemical cleavage of intactimmunoglobulins or they may be genetically engineered by recombinant DNAtechniques. The methods of production are well known in the art and aredescribed, for example, in Antibodies: A Laboratory Manual, Edited by:E. Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., which is incorporated herein by reference. A bindingmolecule or antigen-binding fragment thereof may have one or morebinding sites. If there is more than one binding site, the binding sitesmay be identical to one another or they may be different.

The binding molecule can be a naked or unconjugated binding molecule. Anaked or unconjugated binding molecule is intended to refer to a bindingmolecule that is not conjugated, operatively linked or otherwisephysically or functionally associated with an effector moiety or tag,such as inter alia a toxic substance, a radioactive substance, aliposome, or an enzyme. It will be understood that naked or unconjugatedbinding molecules do not exclude binding molecules that have beenstabilized, multimerized, humanized or in any other way manipulated,other than by the attachment of an effector moiety or tag. Accordingly,all post-translationally modified naked and unconjugated bindingmolecules are included herewith, including where the modifications aremade in the natural binding molecule-producing cell environment, by arecombinant binding molecule-producing cell, and are introduced by thehand of man after initial binding molecule preparation. Of course, theterm naked or unconjugated binding molecule does not exclude the abilityof the binding molecule to form functional associations with effectorcells and/or molecules after administration to the body, as some of suchinteractions are necessary in order to exert a biological effect. Thelack of associated effector group or tag is therefore applied indefinition to the naked or unconjugated binding molecule in vitro, notin vivo.

Complementary Determining Regions (CDR)

The term “complementary determining regions” as used herein meanssequences within the variable regions of binding molecules, such asimmunoglobulins, that generate the antigen binding site which iscomplementary in shape and charge distribution to the epitope recognizedon the antigen. The CDR regions can be specific for linear epitopes,discontinuous epitopes, or conformational epitopes of proteins orprotein fragments, either as present on the protein in its nativeconformation or, in some cases, as present on the proteins as denatured,e.g., by solubilization in SDS. Epitopes may also consist ofposttranslational modifications of proteins.

Deletion

The term “deletion,” as used herein, denotes a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent as compared to the parent,often the naturally occurring, molecule.

Expression-Regulating Nucleic Acid Sequence

The term “expression-regulating nucleic acid sequence” as used hereinrefers to polynucleotide sequences necessary for and/or affecting theexpression of an operably linked coding sequence in a particular hostorganism. Generally, when two nucleic acid sequences are operablylinked, they will be in the same orientation and usually also in thesame reading frame. They usually will be essentially contiguous,although this may not be required. The expression-regulating nucleicacid sequences, such as inter alia appropriate transcription initiation,termination, promoter, enhancer sequences; repressor or activatorsequences; efficient RNA processing signals such as splicing andpolyadenylation signals; sequences that stabilize cytoplasmic mRNA;sequences that enhance translation efficiency (e.g., ribosome bindingsites); sequences that enhance protein stability; and when desired,sequences that enhance protein secretion, can be any nucleic acidsequence showing activity in the host organism of choice and can bederived from genes encoding proteins, which are either homologous orheterologous to the host organism.

Functional Variant

The term “functional variant,” as used herein, refers to a bindingmolecule that comprises a nucleotide and/or amino acid sequence that isaltered by one or more nucleotides and/or amino acids compared to thenucleotide and/or amino acid sequences of the parent binding moleculeand that is still capable of competing for binding to the bindingpartner, e.g. OX40-receptor, with the parent binding molecule. In otherwords, the modifications in the amino acid and/or nucleotide sequence ofthe parent binding molecule do not significantly affect or alter thebinding characteristics of the binding molecule encoded by thenucleotide sequence or containing the amino acid sequence, i.e. thebinding molecule is still able to recognize and bind its target. Thefunctional variant may have conservative sequence modificationsincluding nucleotide and amino acid substitutions, additions anddeletions. These modifications can be introduced by standard techniquesknown in the art, such as site-directed mutagenesis and randomPCR-mediated mutagenesis, and may comprise natural as well asnon-natural nucleotides and amino acids.

Conservative amino acid substitutions include the ones in which theamino acid residue is replaced with an amino acid residue having similarstructural or chemical properties. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cystine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Furthermore, a variant may have non-conservativeamino acid substitutions, e.g., replacement of an amino acid with anamino acid residue having different structural or chemical properties.Similar minor variations may also include amino acid deletions orinsertions, or both. Guidance in determining which amino acid residuesmay be substituted, inserted, or deleted without abolishingimmunological activity may be found using computer programs well knownin the art.

A mutation in a nucleotide sequence can be a single alteration made at alocus (a point mutation), such as transition or transversion mutations,or alternatively, multiple nucleotides may be inserted, deleted orchanged at a single locus. In addition, one or more alterations may bemade at any number of loci within a nucleotide sequence. The mutationsmay be performed by any suitable method known in the art.

Host

The term “host,” as used herein, is intended to refer to an organism ora cell into which a vector such as a cloning vector or an expressionvector has been introduced. The organism or cell can be prokaryotic oreukaryotic. It should be understood that this term is intended to refernot only to the particular subject organism or cell, but to the progenyof such an organism or cell as well. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentorganism or cell, but are still included within the scope of the term“host” as used herein.

Human

The term “human,” when applied to binding molecules as defined herein,refers to molecules that are either directly derived from a human orbased upon a human sequence. When a binding molecule is derived from orbased on a human sequence and subsequently modified, it is still to beconsidered human as used throughout the specification. In other words,the term human, when applied to binding molecules is intended to includebinding molecules having variable and constant regions derived fromhuman germline immunoglobulin sequences based on variable or constantregions either or not occurring in a human or human lymphocyte or inmodified form. Thus, the human binding molecules may include amino acidresidues not encoded by human germline immunoglobulin sequences,comprise substitutions and/or deletions (e.g., mutations introduced byfor instance random or site-specific mutagenesis in vitro or by somaticmutation in vivo). “Based on” as used herein refers to the situationthat a nucleic acid sequence may be exactly copied from a template, orwith minor mutations, such as by error-prone PCR methods, orsynthetically made matching the template exactly or with minormodifications. Semisynthetic molecules based on human sequences are alsoconsidered to be human as used herein.

Immune Response

The term “immune response” as used herein refers to an antagonistic andspecific host reaction in response to foreign or self antigens,involving the formation of antibodies by B-cells or a cell-mediatedresponse by T-cells.

Insertion

The term “insertion,” also known as the term “addition,” denotes achange in an amino acid or nucleotide sequence resulting in the additionof one or more amino acid or nucleotide residues, respectively, ascompared to the parent, often the naturally occurring, molecule.

Internalizing Binding Molecule

The term “internalizing binding molecule” as used herein means a bindingmolecule as defined herein that is capable of being internalized withinthe target cells to which it binds. In other words, the binding moleculeis taken up, i.e. transported from the outside (cell surface) of atarget cell to the inside, e.g. into the endosomal compartment or othercompartment or into the cytoplasm of the cell, by the target cells uponbinding to the binding partner of the binding molecule.

Isolated

The term “isolated,” when applied to binding molecules as definedherein, refers to binding molecules that are substantially free of otherproteins or polypeptides, particularly free of other binding moleculeshaving different antigenic specificities, and are also substantiallyfree of other cellular or tissue material and/or chemical precursors orother chemicals. For example, when the binding molecules arerecombinantly produced, they are preferably substantially free ofculture medium, and when the binding molecules are produced by chemicalsynthesis, they are preferably substantially free of chemical precursorsor other chemicals, i.e., they are separated from chemical precursors orother chemicals which are involved in the synthesis of the protein.Preferably, substantially free means that the binding molecule willtypically comprise about 50%, 60%, 70%, 80% or 90% W/W of a sample, moreusually about 95%, and preferably will be over 99% pure.

The term “isolated” when applied to nucleic acid molecules encodingbinding molecules as defined herein, is intended to refer to nucleicacid molecules in which the nucleotide sequences encoding the bindingmolecules are free of other nucleotide sequences, particularlynucleotide sequences encoding binding molecules that bind bindingpartners other than the OX40-receptor. Furthermore, the term “isolated”refers to nucleic acid molecules that are substantially separated fromother cellular components that naturally accompany the native nucleicacid molecule in its natural host, e.g., ribosomes, polymerases, orgenomic sequences with which it is naturally associated. Moreover,“isolated” nucleic acid molecules, such as a cDNA molecules, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

Monoclonal Antibody

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody displaying a single binding specificity which havevariable and constant regions derived from or based on human germlineimmunoglobulin sequences or derived from completely synthetic sequences.

Naturally Occurring

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organismthat can be isolated from a source in nature and which has not beenintentionally modified by man in the laboratory is naturally-occurring.

Neoplastic Cells

The term “neoplastic cells” as used herein refers to cells that resultfrom abnormal autonomous new growth which has no apparent physiologicalfunction. A neoplastic cell further includes transformed cells andcancer cells including blood cancers (benign and malignant).

Nucleic Acid Molecule

The term “nucleic acid molecule” as used in the present invention refersto a polymeric form of nucleotides and includes both sense and antisensestrands of RNA, cDNA, genomic DNA, and synthetic forms and mixedpolymers of the above. A nucleotide refers to a ribonucleotide,deoxynucleotide or a modified form of either type of nucleotide. Theterm also includes single- and double-stranded forms of DNA. Inaddition, a polynucleotide may include either or bothnaturally-occurring and modified nucleotides linked together bynaturally-occurring and/or non-naturally occurring nucleotide linkages.The nucleic acid molecules may be modified chemically or biochemicallyor may contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). The above term is also intended to include anytopological conformation, including single-stranded, double-stranded,partially duplexed, triplex, hairpinned, circular and padlockedconformations. Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule. A reference to a nucleic acid sequence encompasses itscomplement unless otherwise specified. Thus, a reference to a nucleicacid molecule having a particular sequence should be understood toencompass its complementary strand, with its complementary sequence. Thecomplementary strand is also useful, e.g., for antisense therapy,hybridization probes and PCR primers.

Operably Linked

The term “operably linked” refers to two or more nucleic acid sequenceelements that are physically linked and are in a functional relationshipwith each other. For instance, a promoter is operably linked to a codingsequence if the promoter is able to initiate or regulate thetranscription or expression of a coding sequence, in which case thecoding sequence should be understood as being “under the control of” thepromoter. Generally, when two nucleic acid sequences are operablylinked, they will be in the same orientation and usually also in thesame reading frame. They usually will be essentially contiguous,although this may not be required.

Pharmaceutically Acceptable Excipient

By “pharmaceutically acceptable excipient” is meant any inert substancethat is combined with an active molecule such as a drug, agent, orbinding molecule for preparing an agreeable or convenient dosage form.The “pharmaceutically acceptable excipient” is an excipient that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation comprising thedrug, agent or binding molecule.

Specifically Binding

The term “specifically binding,” as used herein, in reference to theinteraction of a binding molecule, e.g. an antibody, and its bindingpartner, e.g. an antigen, means that the interaction is dependent uponthe presence of a particular structure, e.g. an antigenic determinant orepitope, on the binding partner. In other words, the antibodypreferentially binds or recognizes the binding partner even when thebinding partner is present in a mixture of other molecules. The bindingmay be mediated by covalent or non-covalent interactions or acombination of both. In yet other words, the term “specifically binding”means immunospecifically binding to an antigen or a fragment thereof andnot immunospecifically binding to other antigens. A binding moleculethat immunospecifically binds to an antigen may bind to other peptidesor polypeptides with lower affinity as determined by, e.g.,radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA),BIAcore, or other assays known in the art. Binding molecules orfragments thereof that immunospecifically bind to an antigen may becross-reactive with related antigens. Preferably, binding molecules orfragments thereof that immunospecifically bind to an antigen do notcross-react with other antigens.

Substitutions

A “substitution,” as used herein, denotes the replacement of one or moreamino acids or nucleotides by different amino acids or nucleotides,respectively.

Therapeutically Effective Amount

The term “therapeutically effective amount” refers to an amount of thebinding molecule as defined herein that is effective for preventing,ameliorating or treating a disorder or disease wherein the OX40-receptormolecules play a role or are associated with.

Treatment

The term “treatment” refers to therapeutic treatment as well asprophylactic or preventative measures to cure or halt or at least retarddisease progress. Those in need of treatment include those alreadyinflicted with a disease or disorder wherein OX40-receptor moleculesplay a role or are associated with as well as those in which the diseaseor disorder is to be prevented. Prevention encompasses inhibiting orreducing the spread of the disease or disorder or inhibiting or reducingthe onset, development or progression of one or more of the symptomsassociated with the disease or disorder wherein OX40-receptor moleculesplay a role or are associated with.

Vector

The term “vector” denotes a nucleic acid molecule into which a secondnucleic acid molecule can be inserted for introduction into a host whereit will be replicated, and in some cases expressed. In other words, avector is capable of transporting a nucleic acid molecule to which ithas been linked. Cloning as well as expression vectors are contemplatedby the term “vector,” as used herein. Vectors include, but are notlimited to, plasmids, cosmids, bacterial artificial chromosomes (BAC)and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. A vector containing a second nucleic acidmolecule is introduced into a cell by transformation, transfection, orby making use of viral entry mechanisms. Certain vectors are capable ofautonomous replication in a host into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication). Othervectors can be integrated into the genome of a host upon introductioninto the host, and thereby are replicated along with the host genome.

In a first aspect, the present invention provides agonistic bindingmolecules, capable of binding, preferably specifically binding, to orcapable of associating with the human OX40-receptor. The agonisticbinding molecules are also capable of binding, particularly specificallybinding, to a fragment of the human OX40-receptor, the fragment at leastcomprising an antigenic determinant of the human OX40-receptor that isrecognized by at least one of the agonistic binding molecules of theinvention. The human OX40-receptor is selectively expressed by activatedimmune cells, such as activated CD4+ T-cells. The binding molecules ofthe invention are capable of stimulating and/or activating and/orenhancing and/or augmenting and/or inducing activated CD4+ T-cells. Theexpression of CD4 on activated T-cells can be measured by methods knownin the art, including, but not limited to, FACS analysis,immunofluorescence assays, RT-PCR, Northern blot analysis and Westernblot analysis.

In a preferred embodiment, the agonistic binding molecules according tothe invention are human agonistic binding molecules. Preferably, thehuman binding molecules are derived from a semisynthetic library basedon human sequences and mutated using error-prone PCR to increasespecificities. A human binding molecule according to the invention suchas an antibody lacks murine-derived sequences, in contrast to mouseantibodies obtained by hybridoma technology (Kohler and Milstein, 1975),or variants thereof such as chimeric antibodies or humanized antibodies.Human antibodies have the advantage that when administered to humans ananti-antibody immunogenic response will be extremely low or absent,whereas the murine derived antibodies can give rise to such responsesquite extensively (Van Kroonenburgh and Pauwels, 1988). A bindingmolecule is for instance based upon a human sequence when it has beenobtained from a library of human binding molecules. Such a library mayalso comprise human binding molecules that are based upon a humansequence but containing mutations, e.g. a semi-synthetic library, as wasused to obtain molecules according to the present invention. ‘Basedupon’ as used herein, is meant to include the synthetic construction ofgenetic information based upon knowledge of such genetic information.Such methods include the use of human or human derived genetic materialas a template for PCR to construct a new binding molecule encodingconstruct that is based upon the sequence of the template, theconstruction of completely synthetic genetic information with a desiredsequence e.g. by linking synthetic oligonucleotides to a desiredconstruct, and the like. It is to be understood that ‘based upon’ doesnot exclusively mean a direct cloning of the wild type DNA. A personskilled in the art will also be aware of the possibilities of molecularbiology to obtain mutant forms of a certain piece of nucleic acid.

The agonistic binding molecules of the invention can be intactimmunoglobulin molecules such as polyclonal or monoclonal antibodies, inparticular human monoclonal antibodies, or the binding molecules can beantigen-binding fragments including, but not limited to, Fab, F(ab′),F(ab′)2, Fv, dAb, Fd, complementarity determining region (CDR)fragments, single-chain antibodies (scFv), bivalent single-chainantibodies, diabodies, triabodies, tetrabodies, and (poly)peptides thatcontain at least a fragment of an immunoglobulin that is sufficient toconfer specific antigen binding to the (poly)peptides. The agonisticbinding molecules of the invention can be used in non-isolated orisolated form. Furthermore, the agonistic binding molecules of theinvention can be used alone or in a mixture/composition comprising atleast one agonistic binding molecule (or variant or fragment thereof) ofthe invention. The mixture/composition may further comprise at least oneother therapeutic agent. In one embodiment, the therapeutic agent can bea natural ligand of the OX40-receptor or a variant of the natural ligandstill capable of binding to the human OX40-receptor. The agonisticbinding molecules of the invention can act synergistically in vitro withthe natural ligand, e.g. OX40-ligand. An advantage of agonistic bindingmolecules acting synergistically with the natural ligand could be thatthey may enhance the effect of OX40-ligand present in vivo, rather thanonly substituting it. Such synergistic activity can be determined by afunctional assays known to the skilled artisan.

Typically, agonistic binding molecules according to the invention canbind to their binding partners, i.e. the human OX40-receptor, with anaffinity constant (Kd-value) that is lower than 0.2×10⁻⁴ M, 1.0×10⁻⁵ M,1.0×10⁻⁶ M, 1.0×10⁻⁶ M, preferably lower than 1.0×10⁻⁸ M, morepreferably lower than 1.0×10⁻⁹ M, more preferably lower than 1.0×10⁻¹⁰M, even more preferably lower than 1.0×10⁻¹¹ M, and in particular lowerthan 1.0×10⁻¹² M. The affinity constants can vary for antibody isotypes.For example, affinity binding for an IgM isotype refers to a bindingaffinity of at least about 1.0×10⁻⁷ M. Affinity constants can bemeasured using surface plasmon resonance, i.e. an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore system (Pharmacia Biosensor AB,Uppsala, Sweden).

The agonistic binding molecule of the invention may internalize uponbinding to the human OX40-receptor. Furthermore, the agonistic bindingmolecules according to the invention may bind to the human OX40-receptorin soluble form or may bind to the human OX40-receptor bound or attachedto a carrier or substrate, e.g., microtiter plates, membranes and beads,etc. Carriers or substrates may be made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon, nitrocellulose, or teflon, etc.The surface of such supports may be solid or porous and of anyconvenient shape. Furthermore, the agonistic binding molecules may bindto the human OX40-receptor in purified or non-purified form. Preferably,the agonistic binding molecules are capable of specifically binding tothe human OX40-receptor associated with cells, such as activated CD4+T-cells or portions or parts of these cells comprising the humanOX40-receptor or a fragment thereof.

In another embodiment, the binding molecules of the invention comprisesat least a CDR3 region comprising the amino acid sequence selected fromthe group consisting of SEQ ID NO:17 (DRYSQVHYALDY), SEQ ID NO:18(DRYVNTSNAFDY), SEQ ID NO:19 (DMSGFHEFDY), SEQ ID NO:20 (DRYFRQQNAFDY),SEQ ID NO:21 (ARAAGTIFDY), SEQ ID NO:22 (DRYITLPNALDY), SEQ ID NO:23(YDEPLTIYWFDS) and SEQ ID NO:24 (YDNVMGLYWFDY).

In yet another aspect, the invention provides binding molecules of theinvention comprising a heavy chain comprising the amino acid sequenceselected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27 and SEQ ID NO:28. In a further embodiment the invention pertainsto binding molecules comprising a heavy chain comprising the amino acidsequence of SEQ ID NO:25 and a light chain comprising the amino acidsequence of SEQ ID NO:29, a heavy chain comprising the amino acidsequence of SEQ ID NO:26 and a light chain comprising the amino acidsequence of SEQ ID NO:30, a heavy chain comprising the amino acidsequence of SEQ ID NO:27 and a light chain comprising the amino acidsequence of SEQ ID NO:31 or a heavy chain comprising the amino acidsequence of SEQ ID NO:28 and a light chain comprising the amino acidsequence of SEQ ID NO:32.

Another aspect of the invention includes functional variants ofagonistic binding molecules or fragments thereof as defined herein.Molecules are functional variants of a binding molecule, when thevariants are capable of competing for specifically binding to the humanOX40-receptor, preferably competing for the same binding site on thehuman OX40-receptor, with the parent binding molecules. In other words,when the functional variants are still capable of immunospecificallybinding to the human OX40-receptor or a portion thereof. Furthermore,the functional variants must be capable ofinducing/stimulating/enhancing/augmenting activated CD4+ T-cells. Inother words, the functional variants must also have agonistic activity.This agonistic activity can be higher or lower than the agonisticactivity of the parent binding molecules of the invention. Functionalvariants include, but are not limited to, derivatives that aresubstantially similar in primary structural sequence, but which containe.g. in vitro or in vivo modifications, chemical and/or biochemical,that are not found in the parent binding molecule. Such modificationsinclude inter alia acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Alternatively, functional variants can be binding molecules as definedin the present invention comprising an amino acid sequence containingsubstitutions, insertions, deletions or combinations thereof of one ormore amino acids compared to the amino acid sequences of the parentbinding molecules. Furthermore, functional variants can comprisetruncations of the amino acid sequence at either or both the amino orcarboxy termini. Functional variants according to the invention may havethe same or different, either higher or lower, binding affinitiescompared to the parent binding molecule but are still capable of bindingto the human OX40-receptor present on e.g. a CD4+ T-cell. For instance,functional variants according to the invention may have increased ordecreased binding affinities for the human OX40-receptor compared to theparent binding molecules. Preferably, the amino acid sequences of thevariable regions, including, but not limited to, framework regions,hypervariable regions, in particular the CDR3 regions, are modified.Generally, the light chain and the heavy chain variable regions comprisethree hypervariable regions, comprising three CDRs, and more conservedregions, the so-called framework regions (FRs). The hypervariableregions comprise amino acid residues from CDRs and amino acid residuesfrom hypervariable loops. Functional variants intended to fall withinthe scope of the present invention have at least 50%, preferably atleast 60%, at least 70%, at least 75%, more preferably at least 80%, atleast 85%, even more preferably at least 90%, at least 95%, and inparticluar at least 97%, at least 98%, at least 99% amino acid sequencehomology with the parent binding molecules as defined herein. Computeralgorithms such as inter alia Gap or Bestfit known to a person skilledin the art can be used to optimally align amino acid sequences to becompared and to define similar or identical amino acid residues.

Functional variants of the invention can be obtained by altering thenucleotide sequence of parent binding molecules or parts thereof bygeneral molecular biology methods known in the art including, but notlimited to, error-prone PCR, oligonucleotide-directed mutagenesis andsite-directed mutagenesis. Mutations in the nucleotide sequences mayrender a different functionality, but they may also be silent in a waythat certain mutations do not alter the functionality of that particularpiece of DNA and its encoded protein. A person skilled in the art willappreciate the fact that certain deletions, swaps, (point)mutations,additions, substitutions etc. may still result in a nucleic acid thathas a similar function as the original nucleic acid. It is therefore tobe understood that such alterations that do not significantly alter thefunctionality of the encoded agonistic binding molecules against thehuman OX40-receptor are within the scope of the present invention. Humanantibodies according to the invention may therefore also contain(semi-)synthetic regions, e.g. in the CDR regions. It is for instancepossible to alter the CDR regions of the variable domains of bindingmolecules by site-directed mutagenesis, oligo-directed mutagenesis,error-prone PCR, cloning of restriction fragments, and the like.

In yet a further aspect, the invention includes immunoconjugates, i.e.molecules comprising at least one agonistic binding molecule as definedherein and further comprising at least one tag, such as a therapeuticmoiety. Also contemplated in the present invention are mixtures ofimmunoconjugates according to the invention or mixtures of at least oneimmunoconjugates according to the invention and another molecule, suchas a therapeutic agent or another binding molecule. In an embodiment,the immunoconjugates of the invention comprise more than one tag. Thesetags can be the same or distinct from each other and can bejoined/conjugated non-covalently to the binding molecules. The tags canbe joined/conjugated directly to the binding molecules through covalentbonding, including, but not limited to, disulfide bonding, hydrogenbonding, electrostatic bonding, recombinant fusion and conformationalbonding. Alternatively, the tags can be joined/conjugated to the bindingmolecules by means of one or more linking compounds. Techniques forconjugating tags to binding molecules, are well known, see, e.g., Arnonet al., Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy, p. 243-256 in Monoclonal Antibodies And Cancer Therapy (1985),Edited by: Reisfeld et al., A. R. Liss, Inc.; Hellstrom et al.,Antibodies For Drug Delivery, p. 623-653 in Controlled Drug Delivery,2^(nd) edition (1987), Edited by: Robinson et al., Marcel Dekker, Inc.;Thorpe, Antibody Carriers Of Cytotoxic Agents, p. 475-506 In CancerTherapy: A Review, in Monoclonal Antibodies'84 : Biological And ClinicalApplications (1985), Edited by: Pinchera et al.; Analysis, Results, AndFuture Prospective Of The Therapeutic Use Of Radiolabeled Antibody InCancer Therapy, p. 303-316 in Monoclonal Antibodies For Cancer DetectionAnd Therapy (1985), Edited by: Baldwin et al., Academic Press.

In a specific embodiment, the tags comprise a compound that furtherenhances the immune response, such as a compound that stimulates and/oractivates and/or enhances and/or augments and/or induces activatedimmune cells, e.g. activated T-cells such as activated CD4+ T-cells.Such compounds may include, but are not limited to, binding molecules,small molecules, organic or inorganic compounds, enzymes, polynucleotidesequences, plasmids, proteins, peptides, liposomes or combinationsthereof. Examples of compounds capable of enhancing the immune responseinclude, but are not limited to, compounds that activate a cytokinereceptor such as inter alia cytokines including, but not limited to,CSF-1, Flt3 ligand, G-CSF, GM-CSF, IFN-α, IFN-βIFN-γ, IL-1β, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-18, M-CSF,and TNF-α; chemokines including, but not limited to, IP-10, MIG, andMIP-1; binding molecules that immunospecifically bind to a receptorincluding, but not limited to, the CSF-1 receptor, Flt3, G-CSF receptor,GM-CSF receptor, IFN-α receptor, IFN-β receptor, IFN-γ receptor, IL-1βreceptor, IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor,IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor, IL-10receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IP-10receptor, M-CSF receptor, MIG receptor, MT-1 receptor, and TNF-αreceptor. Analogs, derivatives or fragments of the above listedcompounds which are still functional, i.e. are capable of stimulatingand/or activating and/or enhancing and/or augmenting and/or inducingactivated immune cells, e.g. activated T-cells such as activated CD4+T-cells, can also be used as tags of the invention.

Fusion proteins comprising compounds capable of enhancing the immuneresponse and agonistic binding molecules of the invention can beproduced by methods known in the art such as, e.g., recombinantly byconstructing nucleic acid molecules comprising nucleotide sequencesencoding the agonistic binding molecules in frame with nucleotidesequences encoding the suitable compounds and then expressing thenucleic acid molecules. Alternatively, fusion proteins can be producedchemically by conjugating, directly or indirectly via for instance alinker, agonistic binding molecules as defined herein to a suitablecompound.

Alternatively, the binding molecules as described in the presentinvention can be conjugated to tags and be used for detection and/oranalytical and/or diagnostic purposes. The tags used to label thebinding molecules for those purposes depend on the specificdetection/analysis/diagnosis techniques and/or methods used such asinter alia immunohistochemical staining of tissue samples, flowcytometric detection, scanning laser cytometric detection, fluorescentimmunoassays, enzyme-linked immunosorbent assays (ELISA's),radioimmunoassays (RIA's), bioassays (e.g., growth inhibition assays),Western blotting applications, etc. For immunohistochemical staining oftissue samples preferred labels are enzymes that catalyze production andlocal deposition of a detectable product. Enzymes typically conjugatedto binding molecules to permit their immunohistochemical visualizationare well-known and include, but are not limited to, alkalinephosphatase, P-galactosidase, glucose oxidase, horseradish peroxidase,and urease. Typical substrates for production and deposition of visuallydetectable products include, but are not limited to,o-nitrophenyl-beta-D-galactopyranoside (ONPG), o-phenylenediaminedihydrochloride (OPD), p-nitrophenyl phosphate (PNPP),p-nitrophenyl-beta-D-galactopryanoside (PNPG), 3′,3′diaminobenzidine(DAB), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (CN),5-bromo-4-chloro-3-indolyl-phosphate (BCIP), ABTS, BluoGal,iodonitrotetrazolium (INT), nitroblue tetrazolium chloride (NBT),phenazine methosulfate (PMS), phenolphthalein monophosphate (PMP),tetramethyl benzidine (TMB), tetranitroblue tetrazolium (TNBT), X-Gal,X-Gluc, and X-glucoside. Other substrates that can be used to produceproducts for local deposition are luminescent substrates. For example,in the presence of hydrogen peroxide, horseradish peroxidase cancatalyze the oxidation of cyclic diacylhydrazides such as luminol. Nextto that, binding molecules of the invention can also be labeled usingcolloidal gold or they can be labeled with radioisotopes, such as ³³P,³²P, ³⁵S, ³H, and ¹²⁵I. When the binding molecules of the presentinvention are used for flow cytometric detections, scanning lasercytometric detections, or fluorescent immunoassays, they can usefully belabeled with fluorophores. A wide variety of fluorophores useful forfluorescently labeling the binding molecules of the present inventioninclude, but are not limited to, Alexa Fluor and Alexa Fluor dyes,BODIPY dyes, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamineB, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue,rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Cy2,Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein isothiocyanate (FITC),allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyllprotein (PerCP), Texas Red, fluorescence resonance energy tandemfluorophores such as PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-TexasRed, and APC-Cy7. When the binding molecules of the present inventionare used for secondary detection using labeled avidin, streptavidin,captavidin or neutravidin, the binding molecules may be labeled withbiotin.

Next to that, the binding molecules of the invention may be conjugatedto photoactive agents or dyes such as fluorescent and other chromogensor dyes to use the so obtained immunoconjugates in photoradiation,phototherapy, or photodynamic therapy. The photoactive agents or dyesinclude, but are not limited to, photofrin®, synthetic diporphyrins anddichlorins, phthalocyanines with or without metal substituents,chloroaluminum phthalocyanine with or without varying substituents,O-substituted tetraphenyl porphyrins, 3,1-meso tetrakis(o-propionamidophenyl)porphyrin, verdins, purpurins, tin and zinc derivatives ofoctaethylpurpurin, etiopurpurin, hydroporphyrins, bacteriochlorins ofthe tetra(hydroxyphenyl)porphyrin series, chlorins, chlorin e6,mono-1-aspartyl derivative of chlorin e₆, di-1-aspartyl derivative ofchlorin e₆, tin(IV) chlorin e₆, meta-tetrahydroxyphenylchlor-in,benzoporphyrin derivatives, benzoporphyrin monoacid derivatives,tetracyanoethylene adducts of benzoporphyrin, dimethylacetylenedicarboxylate adducts of benzoporphyrin, Diels-Adler adducts,monoacid ring “a” derivative of benzoporphyrin, sulfonated aluminum PC,sulfonated AlPc, disulfonated, tetrasulfonated derivative, sulfonatedaluminum naphthalocyanines, naphthalocyanines with or without metalsubstituents and with or without varying substituents, anthracenediones,anthrapyrazoles, aminoanthraquinone, phenoxazine dyes, phenothiazinederivatives, chalcogenapyrylium dyes, cationic selena andtellurapyrylium derivatives, ring-substituted cationic PC, pheophorbidederivative, naturally occurring porphyrins, hematoporphyrin, ALA-inducedprotoporphyrin DC, endogenous metabolic precursors, 5-aminolevulinicacid benzonaphthoporphyrazines, cationic imminium salts, tetracyclines,lutetium texaphyrin, tin-etio-purpurin, porphycenes,benzophenothiazinium and combinations thereof.

When the immunoconjugates of the invention are used for in vivodiagnostic use, the binding molecules can also be made detectable byconjugation to e.g. magnetic resonance imaging (MRI) contrast agents,including, but not limited to, agents comprising cobalt (II), copper(II), chromium (III), dysprosium (III), erbium (III), gadolinium (III),holmium (III), iron (II), iron (III), manganese (II), neodymium (III),nickel (II), samarium (III), terbium (III), vanadium (II) or ytterbium(III); ultrasound contrast agents; X-ray contrast agents, including, butnot limited to, agents comprising bismuth (III), gold (III), lanthanum(III) or lead (II); or by radioisotopic labeling, including, but notlimited to, agents comprising copper⁶⁷, gallium⁶⁷, gallium⁶¹, indium¹³³,iodine¹²³, iodine¹²⁵, iodine¹³¹, mercury¹⁹⁷, mercury²⁰³, rhenium¹⁸⁶,rhenium¹⁸⁸, rubidium⁹⁷, rubidium¹⁰³, technetium^(99m) or yttrium⁹⁰.

Furthermore, the binding molecules of the invention can also be attachedto solid supports, which are particularly useful for immunoassays orpurification of the binding partner, i.e. the human OX40-receptor. Suchsolid supports might be porous or nonporous, planar or nonplanar andinclude, but are not limited to, glass, cellulose, polyacrylamide,nylon, polystyrene, polyvinyl chloride or polypropylene supports. Thebinding molecules can also for example usefully be conjugated tofiltration media, such as NHS-activated Sepharose or CNBr-activatedSepharose for purposes of immunoaffinity chromatography. They can alsousefully be attached to paramagnetic microspheres, typically bybiotin-streptavidin interaction. The microspheres can be used forisolation of cells that express or display the human OX40-receptor orfragments thereof. As another example, the binding molecules of thepresent invention can usefully be attached to the surface of amicrotiter plate for ELISA.

It is another aspect of the present invention to provide a nucleic acidmolecule encoding at least a binding molecule or functional fragmentthereof according to the invention. Such nucleic acid molecules can beused as intermediates for cloning purposes, e.g. in the process ofaffinity maturation described above. In a preferred embodiment, thenucleic acid molecules are isolated or purified.

The skilled man will appreciate that functional variants of thesenucleic acid molecules are also intended to be a part of the presentinvention. Functional variants are nucleic acid sequences that can bedirectly translated, using the standard genetic code, to provide anamino acid sequence identical to that translated from the parent nucleicacid molecules. Preferably, the nucleic acid molecules encode agonisticbinding molecules comprising a CDR3 region, preferably a heavy chainCDR3 region, comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:17 (DRYSQVHYALDY), SEQ ID NO:18 (DRYVNTSNAFDY),SEQ ID NO:19 (DMSGFHEFDY), SEQ ID NO:20 (DRYFRQQNAFDY), SEQ ID NO:21(ARAAGTIFDY), SEQ ID NO:22 (DRYITLPNALDY), SEQ ID NO:23 (YDEPLTIYWFDS)and SEQ ID NO:24 (YDNVMGLYWFDY). Even more preferably, the nucleic acidmolecules encode agonistic binding molecules comprising a heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. In yetanother embodiment, the nucleic acid molecules encode binding moleculescomprising a heavy chain comprising the amino acid sequence of SEQ IDNO:25 and a light chain comprising the amino acid sequence of SEQ IDNO:29, or they encode a heavy chain comprising the amino acid sequenceof SEQ ID NO:26 and a light chain comprising the amino acid sequence ofSEQ ID NO:30, or they encode a heavy chain comprising the amino acidsequence of SEQ ID NO:27 and a light chain comprising the amino acidsequence of SEQ ID NO:31, or they encode a heavy chain comprising theamino acid sequence of SEQ ID NO:28 and a light chain comprising theamino acid sequence of SEQ ID NO:32. A further aspect of the inventionpertains to nucleic acid molecules comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41 and SEQ ID NO:42. Nucleic acid molecules comprising a heavy chaincomprising the nucleotide sequence of SEQ ID NO:39 and a light chaincomprising the nucleotide sequence of SEQ ID NO:43, a heavy chaincomprising the nucleotide sequence of SEQ ID NO:40 and a light chaincomprising the nucleotide sequence of SEQ ID NO:44, a heavy chaincomprising the nucleotide sequence of SEQ ID NO:41 and a light chaincomprising the nucleotide sequence of SEQ ID NO:45 or a heavy chaincomprising the nucleotide sequence of SEQ ID NO:42 and a light chaincomprising the nucleotide sequence of SEQ ID NO:46 are also a part ofthe present invention.

Another aspect of nucleic acid molecules according to the presentinvention, is their potential for use in gene-therapy or vaccinationapplications. Therefore, in another embodiment of the invention, nucleicacid molecules according to the invention are provided wherein saidnucleic acid molecule is present in a gene delivery vehicle. A ‘genedelivery vehicle’ as used herein refers to an entity that can be used tointroduce nucleic acid molecules into cells, and includes liposomes,recombinant viruses, and the like. Preferred gene therapy vehicles ofthe present invention will generally be viral vectors, such as comprisedwithin a recombinant retrovirus, herpes simplex virus (HSV), adenovirus,adeno-associated virus (AAV), cytomegalovirus (CMV), and the like. Suchapplications of the nucleic acid sequences according to the inventionare included in the present invention. The person skilled in the artwill be aware of the possibilities of recombinant viruses foradministering sequences of interest to cells. The administration of thenucleic acids of the invention to cells can result in an enhanced immuneresponse.

It is another aspect of the invention to provide vectors, i.e. nucleicacid constructs, comprising one or more nucleic acid molecules accordingto the present invention. Vectors can be derived from plasmids such asinter alia F, R1, RP1, Col, pBR322, TOL, Ti, etc; cosmids; phages suchas lambda, lambdoid, M13, Mu, P1, P22, Qβ, T-even, T-odd, T2, T4, T7,etc; plant viruses such as inter alia alfalfa mosaic virus, bromovirus,capillovirus, carlavirus, carmovirus, caulivirus, clostervirus,comovirus, cryptovirus, cucumovirus, dianthovirus, fabavirus, fijivirus,furovirus, geminivirus, hordeivirus, ilarvirus, luteovirus, machlovirus,marafivirus, necrovirus, nepovirus, phytorepvirus, plant rhabdovirus,potexvirus, potyvirus, sobemovirus, tenuivirus, tobamovirus, tobravirus,tomato spotted wilt virus, tombusvirus, tymovirus, etc; or animalviruses such as inter alia adenovirus, arenaviridae, baculoviridae,Birnaviridae, bunyaviridae, calciviridae, cardioviruses, coronaviridae,corticoviridae, cystoviridae, Epstein-Barr virus, enteroviruses,filoviridae, flaviviridae, Foot-and-Mouth disease virus, hepadnaviridae,hepatitis viruses, herpesviridae, immunodeficiency viruses, influenzavirus, inoviridae, iridoviridae, orthomyxoviridae, papovaviruses,paramyxoviridae, parvoviridae, picornaviridae, poliovirus,polydnaviridae, poxviridae, reoviridae, retroviruses, rhabdoviridae,rhinoviruses, Semliki Forest virus, tetraviridae, togaviridae,toroviridae, vaccinia virus, vescular stomatitis virus, etc. Vectors canbe used for cloning and/or for expression of the agonistic bindingmolecules of the invention and might even be used for gene therapypurposes. Vectors comprising one or more nucleic acid moleculesaccording to the invention operably linked to one or moreexpression-regulating nucleic acid molecules are also covered by thepresent invention. The choice of vector is dependent on the recombinantprocedures followed and the host used. Introduction of vectors in hostcells can be effected by inter alia calcium phosphate transfection,virus infection, DEAE-dextran mediated transfection, lipofectamintransfection or electroporation. Vectors may be autonomously replicatingor may replicate together with the chromosome into which they have beenintegrated. Preferably, the vectors contain one or more selectionmarkers. Useful markers are dependent on the host cells of choice andare well known to persons skilled in the art. They include, but are notlimited to, kanamycin, neomycin, puromycin, hygromycin, zeocin,thymidine kinase gene from Herpes simplex virus (HSV-TK), dihydrofolatereductase gene from mouse (dhfr). Vectors comprising one or more nucleicacid molecules encoding the agonistic binding molecules as describedabove operably linked to one or more nucleic acid molecules encodingproteins or peptides that can be used to isolate the binding moleculesare also covered by the invention. These proteins or peptides include,but are not limited to, glutathione-S-transferase, maltose bindingprotein, metal-binding polyhistidine, green fluorescent protein,luciferase and beta-galactosidase.

Hosts containing one or more copies of the vectors mentioned above arean additional subject of the present invention. Preferably, the hostsare host cells. Host cells include, but are not limited to, cells ofmammalian, plant, insect, fungal or bacterial origin. Bacterial cellsinclude, but are not limited to, cells from Gram positive bacteria suchas several species of the genera Bacillus, Streptomyces andStaphylococcus or cells of Gram negative bacteria such as severalspecies of the genera Escherichia and Pseudomonas. In the group offungal cells preferably yeast cells are used. Expression in yeast can beachieved by using yeast strains such as inter alia Pichia pastoris,Saccharomyces cerevisiae and Hansenula polymorpha. Furthermore, insectcells such as cells from Drosophila and Sf9 can be used as host cells.Besides that, the host cells can be plant cells such as inter alia cellsfrom crop plants such as forestry plants, or cells from plants providingfood and raw materials such as cereal plants, or medicinal plants, orcells from ornamentals, or cells from flower bulb crops. Transformed(transgenic) plants or plant cells are produced by known methods, forexample, Agrobacterium-mediated gene transfer, transformation of leafdiscs, protoplast transformation by polyethylene glycol-induced DNAtransfer, electroporation, sonication, microinjection or bolistic genetransfer. Additionally, a suitable expression system can be abaculovirus system. Expression systems using mammalian cells such asChinese Hamster Ovary (CHO) cells, COS cells, BHK cells or Bowesmelanoma cells are preferred in the present invention. Mammalian cellsprovide expressed proteins with posttranslational modifications that aremost similar to natural molecules of mammalian origin. Since the presentinvention deals with molecules that may have to be administered tohumans, a completely human expression system would be particularlypreferred. Therefore, even more preferably, the host cells are humancells, such as HeLa, 911, AT1080, A549, 293 or PER.C6™ cells (PER.C6 isa trademark owned by Crucell Holland B.V.) and cells derived therefromby genetic modification with antibody encoding expression constructs. Inpreferred embodiments, the producing human cells comprise at least afunctional part of a nucleic acid sequence encoding an adenovirus Elregion in expressible format. In even more preferred embodiments, saidhost cells are derived from a human retina and immortalized with nucleicacids comprising adenoviral El sequences, such as PER.C6™ cells andderivatives thereof. Production of recombinant proteins in host cellscan be performed according to methods well known in the art. The use ofPER.C6™ cells as a production platform for proteins of interest has beendescribed in WO 00/63403 the disclosure of which is incorporated hereinby reference.

It is another aspect of the invention to provide a method of producingagonistic binding molecules or functional variants thereof, preferablyhuman agonistic binding molecules or functional variants thereofaccording to the present invention. The method comprises the steps of a)culturing a host as described above under conditions conducive to theexpression of the agonistic binding molecules, and b) optionally,recovering the expressed agonistic binding molecules. The expressedagonistic binding molecules can be recovered from the cell free extract,but preferably they are recovered from the culture medium. Methods torecover proteins, such as binding molecules, from cell free extracts orculture medium are well known to the man skilled in the art. Agonisticbinding molecules or functional variants thereof as obtainable by theabove described method are also a part of the present invention.

Alternatively, next to the expression in hosts, such as host cells, theagonistic binding molecules of the invention or functional fragmentsthereof can be produced synthetically by conventional peptidesynthesizers or in cell-free translation systems using RNA's derivedfrom DNA molecules according to the invention. Agonistic bindingmolecule as obtainable by the above described synthetic productionmethods or cell-free translation systems are also a part of the presentinvention.

In yet another alternative embodiment, binding molecules according tothe present invention, preferably human agonistic binding moleculesspecifically binding to the human OX40-receptor or fragments thereof,may be generated by transgenic non-human mammals, such as for instancetransgenic mice or rabbits, that express human immunoglobulin genes.Preferably, the transgenic non-human mammals have a genome comprising ahuman heavy chain transgene and a human light chain transgene encodingall or a portion of the human agonistic binding molecules as describedabove. The transgenic non-human mammals can be immunized with a purifiedor enriched preparation of the human OX40-receptor or fragment thereofand/or cells expressing the human OX40-receptor. Protocols forimmunizing non-human mammals are well established in the art. See UsingAntibodies: A Laboratory Manual, Edited by: E. Harlow, D. Lane (1998),Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and CurrentProtocols in Immunology, Edited by: J. E. Coligan, A. M. Kruisbeek, D.H. Margulies, E. M. Shevach, W. Strober (2001), John Wiley & Sons Inc.,New York, the disclosures of which are incorporated herein by reference.Immunization protocols often include multiple immunizations, either withor without adjuvants such as Freund's complete adjuvant and Freund'sincomplete adjuvant, but may also include naked DNA immunizations. Inanother embodiment, the human agonistic binding molecules are producedby B cells or plasma cells derived from the transgenic animals. In yetanother embodiment, the human agonistic binding molecules are producedby hybridomas which are prepared by fusion of B cells obtained from theabove described transgenic non-human mammals to immortalized cells. Bcells, plasma cells and hybridomas as obtainable from the abovedescribed transgenic non-human mammals and human agonistic bindingmolecules as obtainable from the above described transgenic non-humanmammals, B cells, plasma cells and hybridomas are also a part of thepresent invention. In yet another embodiment, human agonistic bindingmolecules of the present invention can also be produced in transgenic,non-human, mammals such as inter alia goats or cows, and can be secretedinto, and optionally recovered from, the milk of the transgenic mammals.

In a further aspect, the invention provides a method of identifyingbinding molecules, preferably human binding molecules such as humanmonoclonal antibodies or fragments thereof, according to the inventionor nucleic acid molecules according to the invention and comprises thesteps of a) contacting a phage library of binding molecules, preferablyhuman binding molecules, with material comprising the humanOX40-receptor or a part thereof, b) selecting at least once for a phagebinding to the material comprising the human OX40-receptor or a partthereof, and c) separating and recovering the phage binding to thematerial comprising the human OX40-receptor or a part thereof. Theselection step according to the present invention is preferablyperformed in the presence of at least part of the human OX40-receptor,e.g. cells transfected with the human OX40-receptor expression plasmids,isolated human OX40-receptor, the extracellular part thereof, fusionproteins comprising such, and the like.

Phage display methods for identifying and obtaining binding molecules,e.g. antibodies, are by now well-established methods known by the personskilled in the art. They are e.g. described in U.S. Pat. No. 5,696,108;Burton and Barbas, 1994; and de Kruif et al., 1995b. For theconstruction of phage display libraries, collections of human monoclonalantibody heavy and light chain variable region genes are expressed onthe surface of bacteriophage, preferably filamentous bacteriophage,particles, in for example single chain Fv (scFv) or in Fab format (seede Kruif et al., 1995b). Large libraries of antibody fragment-expressingphages typically contain more than 1.0×10⁹ antibody specificities andmay be assembled from the immunoglobulin V regions expressed in the Blymphocytes of immunized- or non-immunized individuals. Alternatively,phage display libraries may be constructed from immunoglobulin variableregions that have been partially assembled in vitro to introduceadditional antibody diversity in the library (semi-synthetic libraries).For example, in vitro assembled variable regions contain stretches ofsynthetically produced, randomized or partially randomized DNA in thoseregions of the molecules that are important for antibody specificity,e.g. CDR regions. Antigen specific phage antibodies can be selected fromthe library by immobilizing target antigens such as the humanOX40-receptor or fragments thereof on a solid phase and subsequentlyexposing the target antigens to a phage library to allow binding ofphages expressing antibody fragments specific for the solid phase-boundantigen. Non-bound phages are removed by washing and bound phages elutedfrom the solid phase for infection of Escherichia coli (E. coli)bacteria and subsequent propagation. Multiple rounds of selection andpropagation are usually required to sufficiently enrich for phagesbinding specifically to the target antigen. Phages may also be selectedfor binding to complex antigens such as complex mixtures of proteins orwhole cells such as cells transfected with the human OX40-receptorexpression plasmids or cells naturally expressing the humanOX40-receptor. Selection of antibodies on whole cells has the advantagethat target antigens are presented in their native configuration, i.e.unperturbed by possible conformational changes that might have beenintroduced in the case where an antigen is immobilized to a solid phase.Antigen specific phage antibodies can be selected from the library byincubating a cell population of interest, expressing known and unknownantigens on their surface, with the phage antibody library to let forexample the scFv or Fab part of the phage bind to the antigens on thecell surface. After incubation and several washes to remove unbound andloosely attached phages, the cells of interest are stained with specificfluorescent labeled antibodies and separated on a Fluorescent ActivatedCell Sorter (FACS). Phages that have bound with their scFv or Fab partto these cells are eluted and used to infect Escherichia coli to allowamplification of the new specificity. Generally, one or more selectionrounds are required to separate the phages of interest from the largeexcess of non-binding phages. Monoclonal phage preparations can beanalyzed for their specific staining patterns and allowingidentification of the antigen being recognized (De Kruif et al., 1995a;Lekkerkerker and Logtenberg, 1999). The phage display method can beextended and improved by subtracting non-relevant binders duringscreening by addition of an excess of non-target molecules that aresimilar but not identical to the target, and thereby strongly enhancethe chance of finding relevant binding molecules (see U.S. Pat. No.6,265,150 which is incorporated herewith by reference). In this method,subtraction can be done by the presence of T-cells and other lymphocytesthat do not express the human OX40-receptor.

In yet a further aspect, the invention provides a method of obtaining abinding molecule, preferably a human binding molecule or a nucleic acidmolecule according to the invention, wherein the method comprises thesteps of a) performing the above described method of identifying bindingmolecules, preferably human binding molecules such as human monoclonalantibodies or fragments thereof according to the invention, or nucleicacid molecules according to the invention, and b) isolating from therecovered phage the human binding molecule and/or the nucleic acidencoding the human binding molecule. Once a new monoclonal phageantibody has been established or identified with the above mentionedmethod of identifying binding molecules or nucleic acid moleculesencoding the binding molecules, the DNA encoding the scFv or Fab can beisolated from the bacteria or phages and combined with standardmolecular biological techniques to make constructs encoding bivalentscFv's or complete human immunoglobulins of a desired specificity (e.g.IgG, IgA or IgM). These constructs can be transfected into suitable celllines and complete binding molecules such as human monoclonal antibodiescan be produced (see Huls et al., 1999; Boel et al., 2000).

Preferably, after identifying and obtaining a binding moleculespecifically binding to the human OX40-receptor, it is established ifthe binding molecule has agonistic activity. This can be tested in vitroin a cell culture system or in an animal model system. The cell culturesystem can comprise cells derived from a tissue sample of a patient. Forinstance, activated CD4+ T-cells can be contacted with a bindingmolecule of the invention or a control binding molecule and the abilityof the binding molecule of the invention to enhance the activity ofactivated CD4+ T-cells compared to the control binding molecule can bedetermined. Furthermore, the activation induced by the binding moleculeof the invention might be compared to a well-known inducer of theOX40-receptor such as the OX40-ligand. Moreover, with this kind of testa binding molecule with antagonistic activity can be identified andoptionally used in the treatment of a disorder or disease whereinantagonistic binding molecules capable of binding to the humanOX40-receptor are useful. The ability of binding molecule according tothe invention to modulate (either enhance or decrease) the activity ofactivated CD4+ T-cells can be assessed by detecting the proliferation ofCD4+ T-cells, detecting the activation of signaling molecules, detectingthe effector function of CD4+ T-cells, detecting the expression ofcytokines or antigens, or detecting the differentiation of CD4+ T-cells.Furthermore, agonistic activity of the binding molecules can beestablished by a costimulation assay with for instance the OX40-ligandas described in the present examples. Techniques known to those of skillin the art can be used for measuring these activities. For example,cellular proliferation can be assayed by ³H-thymidine incorporationassays and trypan blue cell counts. The activation of signalingmolecules can be assayed, for example, by kinase assays andelectromobility shift assays (EMSAs). The effector function of T-cellscan be measured, for example, by cytokine ELISA assays or elispotassays. Cytokine and antigen expression can be assayed, for example, byimmunoassays including, but not limited to, competitive andnon-competitive assay systems using techniques such as Western blots,immunohistochemistry, radioimmunoassays, ELISA, sandwich immunoassays,immunoprecipitation assays, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays and FACS analysis. Thebinding molecules of the invention can also be tested in suitable animalmodel systems prior to use in humans. Such animal model systems include,but are not limited to, mice, rats, chicken, cows, monkeys, pigs, dogs,rabbits, etc. Any animal system well-known in the art may be used.

In a further aspect, the invention provides compositions comprising atleast one agonistic binding molecule, at least one functional variant orfragment thereof, at least one immunoconjugate according to theinvention or a combination thereof In addition to that, the compositionsmay comprise inter alia stabilizing molecules, such as albumin orpolyethylene glycol, or salts. Preferably, the salts used are salts thatretain the desired biological activity of the binding molecules and donot impart any undesired toxicological effects. Examples of such saltsinclude, but are not limited to, acid addition salts and base additionsalts. Acid addition salts include, but are not limited to, thosederived from nontoxic inorganic acids, such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydro-iodic, and phosphorous acidsand the like, as well as from nontoxic organic acids such as aliphaticmono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include, but are not limited to, thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like. If necessary, the binding molecules of the invention may becoated in or on a material to protect them from the action of acids orother natural or non-natural conditions that may inactivate the bindingmolecules.

In yet a further aspect, the invention provides compositions comprisingat least one nucleic acid molecule as defined in the present invention.The compositions may comprise aqueous solutions such as aqueoussolutions containing salts (e.g., NaCl or salts as described above),detergents (e.g., SDS) and/or other suitable components.

Furthermore, the present invention pertains to pharmaceuticalcompositions comprising at least one agonistic binding moleculeaccording to the invention, at least one functional variant or fragmentthereof, at least one immunoconjugate according to the invention, atleast one composition according to the invention, or combinationsthereof The pharmaceutical composition of the invention furthercomprises at least one pharmaceutically acceptable excipient. Apharmaceutical composition according to the invention can furthercomprise at least one other therapeutic, prophylactic and/or diagnosticagent. Alternatively, the further therapeutic, prophylactic and/ordiagnostic agents can also be administered separately from thepharmaceutical composition of the invention. The pharmaceuticalcompositions of the invention can be used in vitro, ex vivo or in vivo.Therapeutic agents and prophylactic agents can include, but are notlimited to, toxic substances, radioactive substances, liposomes, bindingmolecules (with or without tags) specifically binding to cancer cellantigens, enzymes, polynucleotide sequences, plasmids, proteins,peptides or combinations thereof Toxic substances include, but are notlimited to, cytotoxic agents, such as small molecule toxins orchemotherapeutic agents, or enzymatically active toxins of bacterial,fungal, plant or animal origin, or fragments thereof In general,suitable chemotherapeutic agents are described in Remington'sPharmaceutical Sciences, 18^(th) edition (1990), Edited by: A. R.Gennaro, Mack Publishing Co., Philadelphia and in Goodman and Gilman'sThe Pharmacological Basis of Therapeutics, 7th edition (1985), Editedby: A. G. Gilman, L. S. Goodman, T. W. Rall and F. Murad. MacMillanPublishing Co., New York. Suitable chemotherapeutic agents that arestill in the experimental phase are known to those of skill in the artand might also be used as toxic substances in the present invention. Ina specific embodiment, therapeutic agents and prophylactic agents caninclude, but are not limited to, compounds that stimulate and/oractivate and/or enhance and/or augment and/or induce activated immunecells, e.g. activated T-cells such as activated CD4+ T-cells. Suchcompounds may include, but are not limited to, binding molecules, smallmolecules, organic or inorganic compounds, enzymes, polynucleotidesequences, plasmids, proteins, peptides, liposomes or combinationsthereof Examples of compounds capable of enhancing the immune responseinclude, but are not limited to, compounds that activate a cytokinereceptor such as inter alia cytokines including, but not limited to,CSF-1, Flt3 ligand, G-CSF, GM-CSF, IFN-α, IFN-β, IFN-γ, IL-1β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-18,M-CSF, and TNF-α; chemokines including, but not limited to, IP-10, MIG,and MIP-1; binding molecules that immunospecifically bind to the CSF-1receptor, Flt3, G-CSF receptor, GM-CSF receptor, IFN-α receptor, IFN-βreceptor, IFN-γ receptor, IL-1β receptor, IL-2 receptor, IL-3 receptor,IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8receptor, IL-9 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor,IL-18 receptor, IP-10 receptor, M-CSF receptor, MIG receptor, MIP-1receptor, and TNF-α receptor. Pharmaceutically acceptable salts, acidsor derivatives, analogs, derivatives or fragments of the above listedcompounds which are still functional, i.e. are capable of stimulatingand/or activating and/or enhancing and/or augmenting and/or inducingactivated immune cells, e.g. activated T-cells such as activated CD4+T-cells, can also be used as further therapeutic or prophylactic agents.In a specific embodiment of the invention the pharmaceutical compositionof the invention comprises an OX40-ligand, preferably a humanOX40-ligand. This ligand can also be administered separately, eitherbefore, subsequent to, or after administration of the pharmaceuticalcomposition of the invention.

Alternatively, the further therapeutic or prophylactic agents include,but are not limited to, anti-viral, anti-microbial, such asanti-bacterial, or anti-fungal agents. Such agents can be bindingmolecules, small molecules, organic or inorganic compounds, enzymes,polynucleotide sequences etc. Examples of anti-microbial agents include,but are not limited to, amifloxacin, amikacin, amoxicillin, amphotericinB, ampicillin, azlocillin, aztreonam, bacampicillin, bacitracin,bifonazole, cafamandole, candicidin, carbenicillin, carbenicillinindanyl, cefaclor, cefadroxil, cefazolin, cefepime, cefonicid,cefoparazone, ceforanide, cefotaxime, cefotetan, cefoxitin, cefpodoximeproxetil, ceftazidime, ceftizoxime, ceftriaxone, cefuroxime, cefuroximeaxetil, cepalospolin, cephadrine, cephalexin, cephalothin,chlortetracycline, cinoxacin, ciprofloxacin, clavulanic acid,cloxacillin, clotrimazole, demeclocycline, dicloxacillin, doxycycline,econazole, erythromycin, fleroxacin, floxacillin, 5-fluorocytosine,fluconazole, gentamicin, griseofulvin, haloprogin, imipenem,itraconazole, kanamycin, ketoconazole, lomefloxacin, loracarbef,methacycline, methicillin, metronidazole, mezlocillin, miconazole,minocycline, moxalactam, nafcillin, natamycin, neomycin, netilmicin,norfloxacin, nystatin, ofloxacin, oxacillin, oxytetracycline,para-aminobenzoic acid, pefloxacin, penicillin G, penicillin V,pentamidine, piperacillin, sparfloxacin, streptomycin, sulfacetamide,sulfadiazine, sulfamethoxazole, sulfanilamide, sulfisoxazole,tetracycline, ticarcillin, tobramycin, trimethoprim-sulfamethoxazolenalidixic acid, vancomycin, vibunazole, and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

Examples of anti-viral agents include, but are not limited to, abacavir,acyclovir, adefovir, afovirsen, amantadine, amprenavir, AZT,camptothecins, castanospermine, cidofovir, D4T, ddC, ddI, d4T,delavirdine, didanosine, efavirenz, famciclovir, fialuridine, foscarnet,FTC, ganciclovir, GG167, idoxuridine, indinavir, interferon alpha,lamivudine, lobucavir, loviride, nelfinavir, nevirapine, oseltamivir,penciclovir, pirodavir, ribavirin, rimantadine, ritonavir, saquinavir,sICAM-1, sorivudine, stavudine, trifluridine, 3TC, valacyclovir,vidarabine, zalcitabine, zanamivir, zidovudine, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Examples of anti-fungal agents include, but are not limited to,amphotericin B, benzoic acid, butoconazole, caprylic acid, ciclopiroxolamine, clotrimazole, econazole, fluconazole, flucytosine,griseofulvin, haloprogin, imidazoles, itraconzole, ketoconazole,miconazole, naftifine, nystatin, potassium iodide, propionic acid,salicyclic acid, terbinafine, terconazole, tolnaftate, and triazoles,undecylenic acid, and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Typically, pharmaceutical compositions must be sterile and stable underthe conditions of manufacture and storage. The agonistic bindingmolecules, variant or fragments thereof, immunoconjugates, nucleic acidmolecules or compositions of the present invention can be in powder formfor reconstitution in the appropriate pharmaceutically acceptableexcipient before or at the time of delivery. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof

Alternatively, the agonistic binding molecules, variant or fragmentsthereof, immunoconjugates, nucleic acid molecules or compositions of thepresent invention can be in solution and the appropriatepharmaceutically acceptable excipient can be added and/or mixed beforeor at the time of delivery to provide a unit dosage injectable form.Preferably, the pharmaceutically acceptable excipient used in thepresent invention is suitable to high drug concentration, can maintainproper fluidity and, if necessary, can delay absorption.

The choice of the optimal route of administration of the pharmaceuticalcompositions will be influenced by several factors including thephysico-chemical properties of the active molecules within thecompositions, the urgency of the clinical situation and the relationshipof the plasma concentrations of the active molecules to the desiredtherapeutic effect. For instance, if necessary, the agonistic bindingmolecules of the invention can be prepared with carriers that willprotect them against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can inter alia be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Furthermore, it may be necessary to coat the agonisticbinding molecules with, or co-administer the agonistic binding moleculeswith, a material or compound that prevents the inactivation of theagonistic binding molecules. For example, the agonistic bindingmolecules may be administered to a subject in an appropriate carrier,for example, liposomes, or a diluent.

The routes of administration can be divided into two main categories,oral and parenteral administration. These two categories include, butare not limited to, bolus, buccal, epidermal, epidural, inhalation,intra-abdominal, intra-arterial, intra-articular, intrabronchial,intracapsular, intracardiac, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebronventricular, intracolic,intracervical, intradermal, intragastric, intrahepatic, intramedullary,intramuscular, intramyocardial, intranasal, intra-ocular intra-orbital,intra-osteal, intrapelvic, intrapericardiac, intraperitoneal,intraplaque, intrapleural, intraprostatic, intrapulmonary, intrarectal,intrarenal, intraretinal, intraspinal, intrasternal, intrasynovial,intrathecal, intrathoracic, intratumoral, intra-uterine, intravenous,intraventricular, intravesical, rectal, spinal, subarachnoid,subcapsular, subcutaneous, subcuticular, sublingual, topical,transdermal, and transmucosal, transtracheal, vaginal administration.

Oral dosage forms can be formulated inter alia as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard capsules, soft gelatin capsules, syrups or elixirs,pills, dragees, liquids, gels, or slurries. These formulations cancontain pharmaceutically acceptable excipients including, but notlimited to, inert diluents such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents such as corn starch or alginic acid; bindingagents such as starch, gelatin or acacia; lubricating agents such ascalcium stearate, glyceryl behenate, hydrogenated vegetable oils,magnesium stearate, mineral oil, polyethylene glycol, sodium stearyl,fumarate, stearic acid, talc, zinc stearate; preservatives such asn-propyl-p-hydroxybenzoate; colouring, flavouring or sweetening agentssuch as sucrose, saccharine, glycerol, propylene glycol or sorbitol;vegetable oils such as arachis oil, olive oil, sesame oil or coconutoil; mineral oils such as liquid parrafin; wetting agents such asbenzalkonium chloride, docusate sodium, lecithin, poloxamer, sodiumlauryl sulfate, sorbitan esters; and thickening agents such as agar,alginic acid, beeswax, carboxymethyl cellulose calcium, carageenan,dextrin or gelatin.

The pharmaceutical compositions of the present invention can also beformulated for parenteral administration. Formulations for parenteraladministration can be inter alia in the form of aqueous or non-aqueousisotonic sterile non-toxic injection or infusion solutions orsuspensions. Preferred parenteral administration routes includeintravenous, intraperitoneal, epidural, intramuscular and intratumoralinjection or infusion. The solutions or suspensions may comprise agentsthat are non-toxic to recipients at the dosages and concentrationsemployed such as 1,3-butanediol, Ringer's solution, Hank's solution,isotonic sodium chloride solution, oils such as synthetic mono- ordiglycerides or fatty acids such as oleic acid, local anesthetic agents,preservatives, buffers, viscosity or solubility increasing agents,water-soluble antioxidants such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like, oil-soluble antioxidants such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like, and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In a further aspect, the invention encompasses the use of an agonisticbinding molecule, a functional variant, an immunoconjugate, a nucleicacid molecule, a composition or a pharmaceutical composition of theinvention for stimulating T-cells, preferably activated CD4+ T-cells invitro. The agonistic binding molecules of the invention can also becontacted together with antigen-presenting cells with T-cells tostimulate T-cell proliferation in vitro.

The agonistic binding molecules, preferably the human agonistic bindingmolecules such as human agonistic monoclonal antibodies according to theinvention, the variants or fragments thereof, the immunoconjugatesaccording to the invention, the nucleic acid molecules according to theinvention, the compositions according to the invention or thepharmaceutical compositions according to the invention can be used asmedicaments. They can inter alia be used in the diagnosis, prophylaxis,treatment, or combination thereof, of a neoplastic, viral, or bacterialdisorder or disease. Preferably, the neoplastic disorder or disease isselected from the group consisting of heavy chain disease, leukemias(e.g., acute myeloid leukemia, chronic myeloid leukemia, chronicmyelomonocytic leukemia, acute promyelocytic leukemia, myelodysplasticsyndrome, juvenile myelomonocytic leukemia, etc.), metastases,neoplasms, tumors (e.g., acoustic neuroma, adenocarcinoma, adrenalcortical cancer, anal carcinoma, angiosarcoma, astrocytoma, basal cellcarcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breastcancer, bronchogenic carcinoma, cancer of the peritoneum, cervicalcancer, chondrosarcoma, chordoma, choriocarcinoma, colon carcinoma,colorectal cancer, craniopharyngioma, cystadenocarcinoma, embryonalcarcinoma, endometrial carcinoma, endotheliosarcoma, ependymoma,epithelial carcinoma, esophageal cancer, Ewing's tumor, fibrosarcoma,gastrointestinal cancer, genitourinary tract cancer , glioblastoma,glioma, head cancer, hemangioblastoma, hepatoma, Hodgkin's Disease,kidney cancer, leiomyosarcoma, liposarcoma, liver cancer, lungcarcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphomas,malignant hypercalcemia, malignant pancreatic insulanoma, medullarycarcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, neckcancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lungcarcinoma, oligodendroglioma, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinomas, papillary carcinoma,penile carcinoma, pinealoma, premalignant skin lesions, primary braintumors, primary macroglobulinemia, primary thrombocytosis, prostatecancer, rectal cancer, renal cell carcinoma, retinoblastoma,rhabdomyosarcoma, salivary gland carcinoma, sarcoma, sebaceous glandcarcinoma, seminoma, small cell lung carcinoma, squamous cell carcinoma,stomach cancer, synovioma, sweat gland carcinoma, testicular tumor,thyroid cancer, uterine carcinoma, vulval cancer, and Wilms tumor), orany disease or disorder characterized by uncontrolled cell growth. Thebinding molecules of the invention are suitable for treatment of yetuntreated patients suffering from any of the above disorders anddiseases, patients who have been or are treated and are in remission orare not in remission, and patients with a recurrent/refractory diseasesor disorders.

Preferably, the viral disorder or disease is selected from the groupconsisting of disorders or diseases associated with the coronavirusresponsible for the Severe Acute Respiratory Syndrome (SARS), herpessimplex virus (HSV), hepatitis B virus (HBV), hepatitis C virus (HCV),human T-cell lymphotrophic virus (HTLV) type I and II, humanimmunodeficiency virus (HIV) type I and II, cytomegalovirus,papillomavirus, polyoma viruses, adenoviruses, Epstein-Barr virus,poxviruses, influenza virus, measles virus, rabies virus, Sendai virus,poliomyelitis virus, coxsackieviruses, rhinoviruses, reoviruses, andrubella virus.

Preferably, the bacterial (for sake of simplicity also including yeastand fungal disorders and diseases) disorder or disease is selected fromthe group consisting of disorders or diseases associated withAcinetobacter sp., Aeromonas hydrophila, Alcaligenes faecalis, Bacilluscereus, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesureolyticus, Bacteroides vulgatus, Borrelia burgdorferi, Borreliavincentii, Brucella abortus, Brucella melitensis, Brucella suis,Campylobacter (Vibrio) fetus, Campylobacter jejuni, Chlamydia spp.,Citrobacter diversus, Citrobacter freundii, Corynebacterium jeikeium,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium ramosum, Clostridium sporogenes, Clostridium sp.,Clostridium tetani, Corynebacterium diphtheriae, Edwardsiella tarda,Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis,Escherichia coli, Francisella tularensis, Haemophilus influenzae,Helicobacter pylori, Klebsiella oxytoca, Klebsiella ozaenae, Klebsiellapneumoniae, Klebsiella rhinoscleromotis, Leptospira icterohemorrhagiae,Mycobacterium tuberculosis, Mycoplasma spp., Neisseria gonorrhoea,Neisseria meningitidis, Peptostreptococcus anaerobius,Peptostreptococcus asaccharolyticus, Peptostreptococcus magnus,Pneumocystis carinii, Prevotella bivia, and Prevotella melaninogenica,Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas stutzeri,Rickettsia prowazeki, Rickettsia tsutsugumushi, Salmonella typhimurium,Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcusaureus, Staphylococcus epidermidis, Streptococcus group C, Streptococcusgroup G, Staphylococcus haemolyticus, Staphylococcus hominis,Staphylococcus simulans, Staphylococcus warneri, Staphylococcus xylosus,Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcusbovis, Streptococcus equinus, Streptococcus pneumoniae, Streptococcuspyogenes, Streptococcus pyogenes, Toxoplasma gondii, Treponemacarateneum, Treponema pallidum, Treponema pertenue, Vibrio cholerae,Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis.

Preferably, the agonistic binding molecules, preferably the humanagonistic binding molecules such as human agonistic monoclonalantibodies according to the invention, the variants or fragmentsthereof, the immunoconjugates according to the invention, the nucleicacid molecules according to the invention, the compositions according tothe invention or the pharmaceutical compositions according to theinvention can be used for enhancing the immune response in a human oranimal, more preferably for enhancing the immune response against atumour, bacterial or viral antigen in a human or animal. In a specificembodiment the agonistic binding molecules of the invention can be usedin combination with the OX40-ligand, preferably the human OX40-ligand.These compounds may exert a costimulatory effect.

As a further aspect, the invention encompasses a method for modulating aT-cell response in a human, comprising the step of administering to saidhuman an effective dose of a binding molecule according to the inventionor a functional variant according to the invention, an immunoconjugateaccording to the invention, a nucleic acid molecule according to theinvention, a vector according to the invention or a pharmaceuticalcomposition according to the invention. Preferably, said modulationcomprises the stimulation of T-cell proliferation.

Another aspect of the invention covers the use of an agonistic bindingmolecule, a functional variant, an immunoconjugate, a nucleic acidmolecule, a composition or a pharmaceutical composition for thepreparation of a medicament for the treatment of a neoplastic, viral orbacterial disorder or a disease as described herein. More preferably,the use will be for the preparation of a medicament for enhancing theimmune response in a human or animal, more preferably the use will befor the preparation of a medicament for enhancing the immune responseagainst a tumour, bacterial or viral antigen in a human or animal.

The molecules are typically formulated in the compositions andpharmaceutical compositions of the invention in a therapeutically,prophylactically or diagnostically effective amount such as for instance1-100 mg/kg, preferably 1-25 mg/kg, more preferably 3-10 mg/kg. Dosageregimens can be adjusted to provide the optimum desired response (e.g.,a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. The molecules and compositionsaccording to the present invention are preferably sterile. Methods torender these molecules and compositions sterile are well known in theart.

Alternatively, cells that are genetically engineered to express thehuman agonistic binding molecules of the invention are administered topatients in vivo. Such cells may be obtained from an animal or patientor an MHC compatible donor and can include, but are not limited tofibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),adipocytes, muscle cells, endothelial cells, etc. The cells aregenetically engineered in vitro using recombinant DNA techniques tointroduce the nucleic acid molecules of the invention into the cells.

Preferably, the agonistic binding molecules are secreted from the cells.The engineered cells which express and preferably secrete the bindingmolecules as described herein can be introduced into the patient forexample systemically, e.g., in the circulation, or intraperitoneally. Inother embodiments, the cells can be incorporated into a matrix or can beencapsulated and implanted in the body.

In another embodiment, activated CD4+ T-cells are removed from a patientand contacted with the agonistic binding molecules of the invention invitro. Thereafter, the treated activated CD4+ T-cells are administeredto the patient. In yet a further specific embodiment, the agonisticbinding molecules of the invention and antigen-presenting cells arecontacted with T-cells to stimulate T-cell proliferation in vitro.

In a specific embodiment, neoplastic, viral or bacterial antigens orcombinations thereof can be administered before, concomitant or afteradministration of the agonistic binding molecules of the invention.Preferably, the antigens are administered to a subject with anneoplastic or infectious disorder or disease prior to (e.g., 2 minutes,5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 7 days, 2 weeks, 4 weeks or 6 weeks before) the administration ofthe agonistic binding molecules of the invention. The antigens include,but are not limited to, recombinantly produced antigens, purifiedantigens, compositions comprising the antigens, neoplastic cellscomprising the antigens, portions of neoplastic cells, such as forinstance membranes, comprising the antigens, fragments of the antigens,viruses, bacteria, fungi, yeast and other microorganisms. If cells areused, the cells can be used directly after removal from the patient, butpreferably the cells are first attenuated before administration to apatient. If viruses or bacteria or other infectious organisms are used,the organisms are preferably first attenuated before administration to apatient. Methods for attenuation are known in the art and include, butare not limited to, irradiation, heat treatment and chemicalinactivation.

In connection with the treatment of neoplastic disorders or diseases,the agonistic binding molecules of the present invention may be used incombination with classical approaches, such as surgery, radiotherapy,chemotherapy, and the like. The human agonistic binding molecules andchemotherapeutic, radiotherapeutic or anti-angiogenic agents may beadministered in a single composition or as two distinct compositionsusing identical or different administration routes. The inventiontherefore also provides combined therapies in which the agonisticbinding molecules of the invention are used simultaneously with, before,or after surgery, radiotherapy or chemotherapy or are administered topatients with, before, or after conventional chemotherapeutic,radiotherapeutic or anti-angiogenic agents. When the administration ofthe human agonistic binding molecule precedes or follows theadministration of the chemotherapeutic, radiotherapeutic oranti-angiogenic agents, intervals ranging from minutes to weeks may liebetween the various administrations. It has to be ensured however that asignificant period of time does not expire between the time of eachdelivery, such that the composition comprising the agent and thecomposition comprising the agonistic binding molecule will still be ableto exert an advantageously combined effect on the neoplasm or tumor. Insuch instances, it is contemplated that one will contact the neoplasm ortumor with both compositions within about 2 minutes to about one week ofeach other and, more preferably, within about 12-72 hours of each other,with a delay time of only about 12-48 hours being most preferred.

In connection with the treatment of viral or bacterial disorders ordiseases as mentioned above, the agonistic binding molecules of thepresent invention may be used in combination with anti-viral and/oranti-bacterial compounds as described above. An similar dosage regimenas indicated for the treatment of neoplastic disorders and diseases canalso be applied for the viral or bacterial disorders or diseases, i.e.one or more human agonistic binding molecules or compositions comprisingthem may be administered to a subject with an infectious disease priorto (e.g., 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeksbefore), concomitantly with, or subsequent to (e.g., 2 minutes, 5minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 7 days, 2 weeks, 4 weeks or 6 weeks after) the administration ofone or more anti-viral and/or anti-bacterial compounds.

Next to that, pharmaceutical packs or kits comprising at least oneagonistic binding molecule, preferably human agonistic binding moleculessuch as human agonistic monoclonal antibodies according to theinvention, at least one variant or fragment thereof, at least oneimmunoconjugate according to the invention, at least one nucleic acidmolecule according to the invention, at least one composition accordingto the invention, at least one pharmaceutical composition according tothe invention, at least one vector according to the invention, at leastone host according to the invention or a combination thereof are also apart of the present invention. Optionally, the kits also contain othertherapeutic or prophylactic compounds. Optionally, the above describedcomponents of the kits of the invention are packed in suitablecontainers and labeled for diagnosis, prophylaxis and/or treatment ofthe indicated conditions. The above-mentioned components may be storedin unit or multi-dose containers, for example, sealed ampules, vials,bottles, syringes, and test tubes, as an aqueous, preferably sterile,solution or as a lyophilized, preferably sterile, formulation forreconstitution. The containers may be formed from a variety of materialssuch as glass or plastic and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The kit mayfurther comprise more containers comprising a pharmaceuticallyacceptable buffer, such as phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, culture medium for one or more ofthe suitable hosts. Associated with the kits can be instructionscustomarily included in commercial packages of therapeutic, prophylacticor diagnostic products, that contain information about for example theindications, usage, dosage, manufacture, administration,contraindications and/or warnings concerning the use of such therapeuticor diagnostic products. The documents providing instructions for the useof the agents of the kit can be in, e.g., written and/or electronicform.

EXAMPLES

To illustrate the invention, the following examples are provided. Theexamples are not intended to limit the scope of the invention in anyway.

Example 1 Selection of Phage Carrying Single Chain Fv FragmentsSpecifically Recognizing Human OX40-Receptor Using OX40-Ig FusionProtein.

Antibody fragments were selected using antibody phage display librariesand MAbstract™ technology, essentially as described in U.S. Pat. No.6,265,150 and in WO 98/15833, both of which are incorporated herein intheir entirety. All procedures were performed at room temperature unlessstated otherwise. A human OX40-Ig fusion protein consisting of theextracellular domain of the human OX40-receptor linked to the CH2 andCH3 domains of human IgG1 was obtained commercially (AlexisBiochemicals) and coated for 2 hours at 37° C. onto the surface ofMaxisorp™ plastic tubes (Nunc) at a concentration of 1.25 μg/ml. Thetubes were blocked for 1 hour in 2% fat free milk powder dissolved inPBS (MPBS). Simultaneously, 500 μl (approximately 10¹³ cfu) of a phagedisplay library expressing single chain Fv fragments (scFv's)essentially prepared as described by De Kruif et al. (1995a) andreferences therein (which are incorporated herein in their entirety),was added to two volumes of 4% MPBS. In addition, human serum was addedto a final concentration of 15% and blocking was allowed to proceed for30-60 minutes. The OX40-Ig-coated tubes were emptied and the blockedphage library was added. The tube was sealed and rotated slowly for 1hour, followed by 2 hours of incubation without rotation. The tubes wereemptied and washed 10 times in PBS containing 0.1% Tween-20, followed bywashing 5 times in PBS. 1 ml glycine-HCL, 0.05 M, pH 2.2 was added, andthe tube was rotated slowly for 10 minutes. The eluted phages were addedto 500 μl 1M Tris-HCl pH 7.4. To this mixture, 3.5 ml of exponentiallygrowing XL-1 blue bacterial culture was added. The tubes were incubatedfor 30 minutes at 37° C. without shaking. Then, the bacteria were platedon 2TY agar plates containing ampicillin, tetracycline and glucose.After overnight incubation of the plates at 37° C., the colonies werescraped from the plates and used to prepare an enriched phage library,essentially as described by De Kruif et al. (1995a). Briefly, scrapedbacteria were used to inoculate 2TY medium containing ampicillin,tetracycline and glucose and grown at a temperature of 37° C. to anOD_(600 nm) of ˜0.3. Helper phages were added and allowed to infect thebacteria after which the medium was changed to 2TY containingampicillin, tetracycline and kanamycin. Incubation was continuedovernight at 30° C. The next day, the bacteria were removed from the 2TYmedium by centrifugation after which the phages were precipitated usingpolyethylene glycol 6000/NaCl. Finally, the phages were dissolved in asmall volume of PBS containing 1% BSA, filter-sterilized and used for anext round of selection. The selection/re-infection procedure wasperformed twice. After the second round of selection, individual E. colicolonies were used to prepare monoclonal phage antibodies. Essentially,individual colonies were grown to log-phase and infected with helperphages after which phage antibody production was allowed to proceedovernight. Phage antibody containing supernatants were tested in ELISAfor binding activity to human OX40-Ig coated 96 wells plates.

Example 2

Validation of the Human OX40R-Specific scFv's.

Selected phage antibodies that were obtained in the screen describedabove, were validated in ELISA for specificity. For this purpose, humanOX40-Ig was coated to Maxisorp™ ELISA plates. After coating, the plateswere blocked in 2% MPBS. The selected phage antibodies were incubated inan equal volume of 4% MPBS. The plates are emptied, washed once in PBS,after which the blocked phages were added. Incubation was allowed toproceed for 1 hour, the plates were washed in PBS containing 0.1%Tween-20 and bound phages were detected using an anti-M13 antibodyconjugated to peroxidase. As a control, the procedure was performedsimultaneously using a control phage antibody directed againstthyroglobulin (De Kruif et al. 1995a and 1995b), which served as anegative control. As shown in FIG. 1, the selected phage antibodiescalled SC02008, SC02009, SC02010, SC02011, SC02012 and SC02021 displayedsignificant binding to the immobilized human OX40-Ig fusion protein.

The phage antibodies that bound to human OX40-Ig were subsequentlytested for binding to human serum IgG to exclude the possibility thatthey recognized the Fc part of the fusion protein. None of the selectedanti-OX40-receptor phages demonstrated binding to human IgG.

In another assay the phage antibodies were analyzed for their ability tobind PER.C6™ cells that recombinantly express human OX40-receptor. Tothis purpose PER.C6™ cells were transfected with a plasmid carrying acDNA sequence encoding human OX40-receptor or with the empty vector andstable transfectants were selected using standard techniques known to aperson skilled in the art (Coligan et al., 2001). For flow cytometryanalysis, phage antibodies were first blocked in an equal volume of 4%MPBS for 15 minutes at 4° C. prior to the staining of the OX40-receptor-and control transfected PER.C6™ cells. The blocked phages were added toa mixture of unlabeled control transfected PER.C6™ cells andOX40-receptor transfected PER.C6™ cells that were labelled green using alipophylic dye (PKH67, Sigma). The binding of the phage antibodies tothe cells was visualized using a biotinylated anti-M13 antibody (SantaCruz Biotechnology) followed by streptavidin-phycoerythrin (Caltag). Asshown in FIG. 2, the selected anti-human OX40-receptor phage antibodiescalled SC02008, SC02009, SC02010, SC02011, SC02012 and SC02021selectively stained the PER.C6™ OX40-receptor transfectant, while theydid not bind the control transfectant.

In another assay the phage antibodies were analyzed for their ability tobind to OX40-receptor positive CD4+ T-cells derived from inflamedtonsils or from synovial fluid from patients suffering from rheumatoidarthritis. As a control, the staining pattern of the anti OX40-receptorphage antibodies on peripheral blood mononuclear cells (MNC) is alsoshown.

Inflamed tonsils were obtained from patients undergoing routinetonsillectomy. Tonsils were minced and the MNC fraction was isolated bydensity centrifugation. Flow cytometric analysis of the binding of theanti-OX40-receptor phage antibodies to the OX40+ CD4+ T-cells wasperformed as described above. The CD4+ T-cells were distinguished fromtotal tonsil MNC using a FITC conjugated antibody against CD4. As shownin FIGS. 3A and 3B the selected anti-human OX40-receptor phageantibodies called SC02008, SC02009, SC02010, SC02011, SC02012 andSC02021 selectively stain a subset of CD4+ T-cells within tonsil andsynovial fluid mononuclear cells respectively, while they display minorstaining of peripheral blood CD4+ T-cells (FIG. 3C).

Example 3 Selection of Phage Carrying Single Chain Fv FragmentsSpecifically Recognizing Human OX40-Receptor Using OX40+ CD4+ T-Cells.

Phage selection experiments were performed as described supra, usinglymphocytes as target. An aliquot of the phage library (500 μl,approximately 10¹³ cfu) were blocked with 2 ml RPMI/10% FCS/1% NHS for15 minutes at room temperature. Tonsil MNC (˜10×10⁶ cells) were added tothe blocked phage library and incubated for 2.5 hours while slowlyrotating at 4° C. Subsequently, the cells were washed twice and wereresuspended in 500 μl RPMI/10% FCS and incubated with a FITC-conjugatedanti-CD4 antibody (Becton Dickinson) and a phycoerythrin-conjugatedanti-OX40-receptor antibody (Becton Dickinson) for 15 minutes on ice.The cells were washed once and transferred to a 4 ml tube. Cell sortingwas performed on a FACSvantage fluorescence-activated cell sorter(Becton Dickinson), and ˜15.000 CD4+ OX40+ cells were sorted. The sortedcells were spun down, the supernatant was saved and the bound phageswere eluted from the cells by resuspending the cells in 500 μl 50 mMglycin pH 2.2 followed by incubation for 5 minutes at room temperature.The mixture was neutralized with 250 μl 1 M Tris-HCl pH 7.4 and added tothe rescued supernatant. Collectively these phages were used to preparean enriched phage library as described earlier. Theselection/re-infection procedure was performed twice. After the secondround of selection, monoclonal phage antibodies were prepared and testedfor binding to tonsillar OX40+ CD4+ T-cells. Selected phage antibodiesthat met this criterium were subsequently tested for binding toOX40-receptor transfected PER.C6™ cells. The results in FIG. 4 show thatthe selected phage antibodies called SC02022 and SC02023 selectivelybind to a subset of CD4+ T-cells within tonsil mononuclear cells (seeFIG. 4A) and that they bind to the human OX40-receptor PER.C6™transfectant (see FIG. 4B).

Example 4

Characterization of the Human OX40-Receptor Specific scFv's.

From the selected human OX40-receptor specific phage antibody (scFv)clones plasmid DNA was obtained and nucleotide sequences were determinedaccording to standard techniques. Nucleotide sequences and amino acidtranslations of the scFv's called SC02008, SC02009, SC02010, SC02011,SC02012, SC02021, SC02022 and SC02023 are shown in FIGS. 5-12,respectively. The nucleotide sequences of the scFv's called SC02008,SC02009, SC02010, SC02011, SC02012, SC02021, SC02022 and SC02023 arealso shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15, respectively. Theamino acid sequences of the scFv's called SC02008, SC02009, SC02010,SC02011, SC02012, SC02021, SC02022 and SC02023 are shown in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14 and SEQ ID NO:16, respectively. The VH and VL gene identity andheavy chain CDR3 compositions of the anti-human OX40-receptor scFv's aredepicted in table 1.

Example 5

Production of Human OX40-Receptor Specific Bivalent scFv in PichiaPastoris.

Methods for the cloning and expression of bivalent scFv fragments in thePichia pastoris system were based on protocols provided by the supplier(Invitrogen) in “A Manual of Methods for Expression of RecombinantProteins Using pPICZ and pPICZα in Pichia pastoris (Version F).” Thebivalent scFv expression vector pPicZbiFVH (see FIG. 13B) wasconstructed from the vector pPICZαB (Invitrogen) (see FIG. 13A)following standard molecular biology techniques known to a personskilled in the art. Three modifications were introduced in the pPICZαB(see FIG. 13C):

-   -   1. A restriction site (NcoI) was introduced by PCR-generated        point mutation directly after the KEK2 cleavage site of the        signal peptide to facilitate cloning into the vector.    -   2. A second NcoI restriction site was removed by PCR generated        point mutation inside the coding region of the sh ble gene.    -   3. A synthetic fragment comprising the hinge region of murine        IgG3 and a linker fragment was introduced between the        restriction sites NotI and XbaI.

All modifications were confirmed by sequencing. ScFv's were cloned intopPicZbiFVH from the phage display expression vector by directionalcloning using the restriction sites NcoI and NotI. The Pichia pastorisstrain SMD1168 kek1:suc1 (ATCC #204414) was transformed with 5-10 μg oflinearized construct cDNA by electroporation according to themanufacturer's protocols (supra). The transformed cells were plated onYPDS agar containing 250 μg/ml Zeocin and incubated at 30° C. for 3-4days. High producing clones were selected by colony lift immunoblotscreening, as follows. Pre-wet nitrocellulose membranes were layeredover the transformation plates and a fraction of each colony was liftedonto the membrane. The membrane was then placed colony side up on YPDagar containing 0.5% methanol and incubated overnight at 30° C. Themembranes were then washed repeatedly with Tris buffered salinecontaining 0.5% Tween-20 (TBST) to removed colonies, then blocked for 30minutes with TBST and 4% non-fat milk powder. The membranes were thenplaced in TBST containing 4% non-fat milk powder and horseradishperoxidase conjugated anti-c-myc antibody (Roche) for 1 hour. Finally,the membranes were washed extensively in TBST followed by a PBS washingstep and scFv-secreting colonies were revealed by a chemofluorescencedetection system (Apbiochem). Selected high producers were purified bystreaking on YPD plates and were subsequently used for bivalent scFvexpression. Small-scale expression cultures were carried out in shakerflasks essentially as described by the manufacturer's protocols (supra).BMGY medium was used for the cell expansion phase, while BMMY medium wasused during the bivalent scFv expression phase. After 48 hours ofinduction supernatants were clarified by repeated centrifugation. Thesupernatant was conditioned for purification by the addition of 1 MNa₂HPO₄ pH 8 to a concentration of 20 mM, 0.5 M Imidazole to aconcentration of 10 mM, 5 M NaCl to a concentration of 500 mM.Hereafter, the samples were purified by immobilized metal affinitychromatography followed by anion exchange chromatography on an AKTAprimeFPLC-system (Pharmacia). A 5 ml HiTrap™ chelating column (Pharmacia) wascharged with NiSO₄ and equilibrated according to the manufacturersinstructions. Conditioned supernatant was loaded directly on to thecolumn and washed extensively in equilibration buffer (20 mM Na₂PO₄ pH8, 10 mM imidazole). Bivalent scFv were eluted directly off the columnon to a 1 ml sepharose Q HP column (Pharmacia) in the presence of 250 mMimidazole pH 8.5. The column was then washed in 20 mM Tris-HCl pH 8,then briefly in 20 mM Na₂PO₄ pH 7.3, and bivalent scFv's were eluted offthe column over a gradient of 0-0.5 M NaCl in 7 column volumes.Fractions were then measured for protein content and were analyzed foractivity and purity. The bivalent scFv clones of SC02008, SC02010,SC02011 and SC02023 were deposited at the European Collection of CellCultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on15 May 2002, under accession numbers 02051563, 02051560, 02051561 and02051562, respectively.

Example 6

Functional Analysis of Bivalent scFv Specifically Recognizing HumanOX40-Receptor.

The anti human-OX40-receptor bivalent scFv's were validated for theirability to bind to OX40+ CD4+ T-cells within tonsil MNC. Tonsil MNCsamples were obtained as described supra and were stained with thebivalent scFv's at a concentration of 5 μg/ml at 4° C. Binding of thebivalent scFv's was visualized using a biotinylated anti-myc antibody(9E10, Santa Cruz Biotechnology) followed by streptavidin-phycoerythrin(Caltag). The bivalent anti human-OX40-receptor scFv's displayed asimilar staining pattern as the corresponding scFv's in phage antibodyformat.

The anti-human OX40-receptor bivalent scFv's were analyzed for theirability to interfere with OX40-receptor-mediated signaling in acostimulation assay. For this purpose 293T cells were transfected witheither the empty vector or with an OX40-ligand cDNA-containing plasmid(pCDNA3.1zeo(+), InVitrogen) using the lipofectamine reagent accordingto standard protocols. 48 hours after transfection, the cells wereharvested, paraformaldehyde fixed and analyzed for cell surfaceexpression of OX40-ligand by flow cytometry (OX40-ligand was visualizedusing the OX40-Ig fusion protein followed by incubation with abiotinylated goat-anti-human Fc polyclonal antibody (Caltag) andstreptavidin-phycoerythrin (Caltag)). To cocultures of 1.5×10³ 293Ttransfectants and 4×10⁵ T-cells, which were activated with asubmitogenic dose of 50 ng/ml of PHA (Abbot Murex), severalconcentrations of the bivalent anti-human-OX40-receptor or controlscFv's were added. T-cells were purified via negative selection usingthe MACS system and a pan-T cell isolation kit (Myltenyi Biotec) fromPBMC that were obtained from healthy donors by Ficoll-Hypaque densitygradients. The cultures were performed in U-bottom 96 well plates for 5days and the proliferation of the T-cells was measured by ³H-thymidineincorporation during the last 16 hours of culture. As shown in FIGS. 14Aand 14B, the bivalent scFv's SC02008 and SC02023, respectively, displayagonistic (stimulating) function in that they induce T-cellproliferation in a concentration dependent manner when incubated withthe mock-transfected 293T cells. Interestingly, these agonistic bivalentanti human-OX40-receptor scFv's demonstrate a synergistic stimulatoryeffect when co-incubated with the OX40-ligand transfected 293T cells ascompared to the level of proliferation that is reached with the sametransfectant in the presence of a control bivalent scFv antibody.

Example 7

Construction of Fully Human Immunoglobulin Molecules from the SelectedAnti-human OX40-Receptor Single Chain Fv Fragments.

To use the selected antibody fragments that recognize humanOX40-receptor for therapeutic applications in humans, it is desirable togenerate human immunoglobulin molecules. The engineering and productionof the human IgG1 monoclonal antibodies is essentially performed asdescribed in detail by Boel et al. (2000). In detail, scFv were reclonedin IgG expression vector C01 (pCRU-KO1). To that purpose, V_(H) andV_(L) regions were PCR amplified using designated primers to appendrestriction sites and restore complete human frameworks. The PCRfragments were cloned in pTOPO (Invitrogen), the integrity of thePCR-fragments was verified by sequencing and thereafter the inserts weresequentially cloned (EcoRI-BamHI for V_(H) and XhoI-NotI for V_(L)) intothe IgG expression vector C01.

ScFv 5′V_(H) oligo 3′V_(H) oligo 5′V_(L) oligo 3′V_(L) oligo 02-008 5H-B3H-B 5K-E 3K-E 02-011 5H-B 3H-B 5K-E 3K-E 02-021 5H-B 3H-B 5K-G 3K-B02-023 5H-B 3H-B 5K-H 3K-F

primer sequences: 5H-B: (SEQ ID NO: 47)acctgtcttgaattctccatggccgaggtgcagctggtggagtctg 3H-B: (SEQ ID NO: 48)gctcgcggatccactcacctgaggagacggtcaccagggtgccctggccc c 5K-E:(SEQ ID NO: 49) acctgtctcgagttttccatggctgacatcgtgatgacacagtctccag 5K-G:(SEQ ID NO: 50) acctgtctcgagttttccatggctgacatcgtgatgacccagtctcc 5K-H:(SEQ ID NO: 51) acctgtctcgagttttccatggctgaaattgtgctcacacagtctccagc cacc3K-E: (SEQ ID NO: 52) ttttccttagcggccgcaaagtgcacttacgtttgatttccagtttggtgccctg 3K-B: (SEQ ID NO: 53)ttttccttagcggccgcaaagtgcacttacgtttgatttccactttggtg ccctg 3K-F:(SEQ ID NO: 54) ttttccttagcggccgcaaagtgcacttacgtttgatctccaccttggtc cctcc

The resulting expression constructs pgG102-008C01, pgG102-011C01,pgG102-021C01 and pgG102-023C01 encoding the human IgG1 antibodiesdirected against human-OX40 receptor were transiently expressed inPER.C6™ cells and supernatants containing IgG1 antibodies were obtained.The expression constructs pgG102-008C01, pgG102-011C01, pgG102-021C01and pgG102-023C01 were deposited at the European Collection of CellCultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on 9June 2003, under provisional accession numbers 03060601, 03060602,03060603 and 03060604, respectively.

The nucleotide sequences of the heavy chains of the antibodies called008, 011, 021 and 023 are shown in SEQ ID NOS:39-42, respectively. Theamino acid sequences of the heavy chains of the antibodies called 008,011, 021 and 023 are shown in SEQ ID NOS:25-28, respectively. Thenucleotide sequences of the light chains of the antibodies called 008,011, 021 and 023 are shown in SEQ ID NOS:43-46, respectively. The aminoacid sequences of the light chains of the antibodies called 008, 011,021 and 023 are shown in SEQ ID NOS:29-32, respectively. Subsequently,the antibodies were purified over size-exclusion columns and protein Acolumns using standard purification methods used generally forimmunoglobulins (see for instance WO 00/63403).

The anti-OX40 receptor IgG1 antibodies were validated for their abilityto bind to PER.C6™ cells transfected with human OX40-receptor. To thispurpose mock- and human OX40-receptor-transfected cells were stainedwith the IgG1 antibodies at a concentration of 20 μg/ml at 4° C. Bindingof the antibodies called 008, 011, 021 and 023 was visualized usingbiotinylated goat-anti-human IgG (Fc specific, Caltag) followed bystreptavidin-phyco-erythrin (Caltag). The stained cells were analyzed byflow cytometry. All antibodies specifically recognized the humanOX40-receptor on OX40-receptor-transfected PER.C6™ cells (filledhistograms in FIG. 15,) while they did not bind the mock-transfectedcell line (open histograms in FIG. 15).

Example 8 Functional Analysis of Fully Human IgG Molecules SpecificallyRecognizing Human OX40-Receptor.

The anti-OX40-receptor IgG1 molecules are validated for their ability tointerfere with OX40R-mediated signaling in a costimulation assay asdescribed supra. It is to be expected that at least one of the IgG1molecules stimulates T-cell proliferation.

Example 9 Immunohistochemistry

The anti-OX40-receptor IgG molecules are biotinylated and subsequentlyanalyzed for their ability to bind to OX40+ cells in inflamed tonsil andtumor sections with infiltrating lymphocytes by immunohistochemistry.Furthermore, they are analyzed for their ability to bind to normaltissues. To this purpose, frozen sections of the following normaltissues: adrenal gland; bladder; brain (cerebellum and cerebrum); bloodvessels (aorta and coronary artery); fallopian tube; oesophagus; stomach(antrum and body); duodenum; ileum; colon; heart; kidney; liver; lung;lymphnode; ovary; pancreas; parathyroid; peripheral nerve; pituitarygland; placenta; prostate; salivary gland; skin; spinal cord; spleen;striated muscle; testis; tonsil; thyroid; ureter and uterus (cervix andendometrium) as well as inflamed tissues and tumor tissues are cut,mounted on glass slides and are dried at room temperature. The sectionsare blocked for endogenous peroxidase with 50 mM sodium azide containing0.03% H₂0₂ for 20 minutes, followed by blocking for endogenous biotinaccording to the provided protocol (X0590, Dako). Subsequently, thesections are blocked with PBS containing 4% BSA and 10% normal humanserum prior to incubation with the biotinylated anti-human OX40 receptorIgG's for 60 minutes at room temperature. To detect bound IgG moleculesthe sections are incubated with streptavidin coupled-horseradishperoxidase (Dako) followed by incubation with diaminobenzidine (Sigma)resulting in a local deposition of brown crystals. The sections arecounterstained with hematoxilin to visualize nucleated cells within thesections. Prior to analysis the sections are dehydrated and the slidesare sealed with eukitt (BDH).

Example 10 In Vivo Analysis of Enhanced Immune Response Induced byAgonistic Anti-Human OX40-Receptor Binding Molecules

To determine the cross-reactivity of the anti-human OX40-receptorantibodies with mouse OX40-receptor, splenic OX40+ CD4+ T-cells areanalyzed by flow cytometry. Murine OX40+ T-cells are generated bystimulating C57B16 splenic CD4 T-cells that are isolated using ananti-CD4-phycoerythrin antibody (Pharmingen) and anti-phycoerythrinlabeled MACS beads (Myltenyi Biotec) with a mitogenic dose of PHA andIL2. The cells are analyzed after 72 hours of stimulation with a ratantibody against the murine OX40-receptor and with the panel ofanti-human OX40-receptor antibodies (supra). In case the agonisticanti-human OX40-receptor antibodies display cross reactivity with mouseOX40-receptor, the OX40-receptor can be engaged in vivo with theseagonistic antibodies to demonstrate the delivery of a costimulatorysignal to effector T-cells. To demonstrate the effect of providing anagonistic anti-OX40-receptor antibody to T-cells during tumor priming invivo, a MCA 303 sarcoma tumor model in C57BL/6 mice is used as describedby Weinberg et al. (2000) and in WO 99/42585. Mice are inoculatedsubcutaneously at day 0 with 1-3×10⁵ MCA 303 sarcoma tumor cells. Threedays later the animals are given intraperitoneal injections with theagonistic anti-human OX40-receptor antibodies at doses ranging from100-500 μg per animal. A second dose is given 7 days after tumorinoculation. The animals are then monitored for tumor growth for over 50days, animals are sacrificed when tumor sizes exceed 1 cubic cm. Whenanimals that are given the agonistic anti-human OX40-receptor antibodiesremain tumor free (or have tumours smaller in size than controlanimals), while animals that are given the tumor cells alone have to besacrificed, this indicates that engagement of the OX40-receptor by theagonistic anti-human OX40-receptor antibodies costimulate effectorT-cells to exert their tumor eradicating function. Alternatively, theexperiment described above can also be performed in a transgenic mousemodel in which human OX40-receptor is expressed under a T-cell specificpromoter. Such a mouse can be created according to protocols known tothe person skilled in the art of transgenic mouse models.

TABLE 1 SEQ ID NO SEQ ID NO of nucleotide of amino acid Name scFvsequence sequence CDR3 V_(H)-germline V_(L)-germline SC02-008 SEQ IDSEQ ID DRYSQVHYAL V_(H)3 DP47 V_(K)II NO: 1 NO: 2 DY (SEQ ID NO: 17)SC02-009 SEQ ID SEQ ID DRYVNTSNAF V_(H)3 DP29 V_(K)II NO: 3 NO: 4 DY(SEQ ID NO: 18) SC02-010 SEQ ID SEQ ID DMSGFHEFDY V_(H)3 DP49 V_(K)INO: 5 NO: 6 (SEQ ID NO: 19) SCO2-011 SEQ ID SEQ ID DRYFRQQNAFV_(H)3 DP47 V_(K)II NO: 7 NO: 8 DY (SEQ ID NO: 20) SCO2-012 SEQ IDSEQ ID ARAAGTIFDY V_(H)3 DP29 V_(K)II NO: 9 NO: 10 (SEQ ID NO: 21)SCO2-021 SEQ ID SEQ ID DRYITLPNALD V_(H)3 DP50 V_(K)II NO: 11 NO: 12 Y(SEQ ID NO: 22) SCO2-022 SEQ ID SEQ ID YDEPLTIYWFD V_(H)3 DP44 V_(K)IIINO: 13 NO: 14 S (SEQ ID NO: 23) SCO2-023 SEQ ID SEQ ID YDNVMGLYWFV_(H)3 DP52 V_(K)II NO: 15 NO: 16 DY (SEQ ID NO: 24)

REFERENCES

-   Al-Shamkhani, A., Birkeland, M. L., Puklavec, M., Brown, M. H.,    James, W., and Barclay, A. N. (1996) OX40 is differentially    expressed on activated rat and mouse T cells and is the sole    receptor for the OX40 ligand. Eur J Immunol 26:1695-1699.-   Boel E, Verlaan S, Poppelier M J, Westerdaal N A, Van Strijp J A and    Logtenberg T (2000) Functional human monoclonal antibodies of all    isotypes constructed from phage display library-derived single-chain    Fv antibody fragments. J Immunol Methods 239:153-166.-   Burton D R and Barbas C F (1994) Human antibodies from combinatorial    libraries. Adv Immunol 57:191-280.-   Coligan J E, Dunn B M, Ploegh H L, Speicher D W and Wingfield P T    (eds.) (2001) Current protocols in protein science, volume I. John    Wiley & Sons, Inc., New York.-   De Kruif J, Terstappen L, Boel E and Logtenberg T (1995a) Rapid    selection of cell subpopulation-specific human monoclonal antibodies    from a synthetic phage antibody library. Proc Natl Acad Sci USA    92:3938.-   De Kruif J, Boel E and Logtenberg T (1995b) Selection and    application of human single chain Fv antibody fragments from a    semi-synthetic phage antibody display library with designed CDR3    regions. J Mol Biol 248:97.-   Huls G, Heijnen I J, Cuomo E, van der Linden J, Boel E, van de    Winkel J and Logtenberg T (1999) Antitumor immune effector    mechanisms recruited by phage display-derived fully human IgG1 and    IgAl monoclonal antibodies. Cancer Res 59: 5778-5784.-   Kaleeba, J. A., Offner, H., Vandenbark, A. A., Lublinski, A., and    Weinberg, A. D. (1998) The OX-40 receptor provides a potent    co-stimulatory signal capable of inducing encephalitogenicity in    myelin-specific CD4+ T cells. Int Immunol 10:453-461.-   Kohler G and Milstein C (1975) Continuous cultures of fused cells    secreting antibody of predefined specificity. Nature 256:495-497.-   Lekkerkerker A and Logtenberg T (1999) Phage antibodies against    human dendritic cell populations obtained by flow cytometry-based    selection on freshly isolated cells. J Immunol Methods 231:53-63.-   Sambrook and Russell (2001) Molecular Cloning, a Laboratory Manual,    third edition. Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y.-   Van Kroonenburgh, M. J., and Pauwels, E. K. (1988) Human    immunological response to mouse monoclonal antibodies in the    treatment or diagnosis of malignant diseases. Nucl Med Commun    9:919-930.-   Weinberg, A. D., Rivera, M. M., Prell, R., Morris, A., Ramstad, T.,    Vetto, J. T., Urba, W. J., Alvord, G., Bunce, C., and    Shields, J. (2000) Engagement of the OX-40 receptor in vivo enhances    antitumor immunity. J Immunol 164:2160-2169.

1-28. (canceled)
 29. A human binding molecule comprising an agonisticbinding molecule capable of binding to and stimulating a humanOX40-receptor, wherein the binding molecule has a synergisticstimulatory effect when co-incubated with OX40-ligand, and wherein thebinding molecule comprises a heavy chain complementary determiningregion 3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:17 and SEQ ID NO:24.
 30. The human bindingmolecule of claim 29, wherein the binding molecule is selected from thegroup consisting of a binding molecule comprising a heavy chain variableregion comprising the variable region of amino acid sequence SEQ IDNO:25, or a sequence that is at least 97% homologous thereto, and alight chain variable region comprising the variable region of amino acidsequence SEQ ID NO: 29, or a sequence that is at least 97% homologousthereto; and a binding molecule comprising a heavy chain variable regioncomprising the variable region of amino acid sequence SEQ ID NO:28, or asequence that is at least 97% homologous thereto, and a light chainvariable region comprising the variable region of amino acid sequenceSEQ ID NO: 32 or a sequence that is at least 97% homologous thereto. 31.The human binding molecule of claim 29, wherein the binding molecule isselected from the group consisting of a binding molecule comprising aheavy chain variable region comprising the variable region of amino acidsequence SEQ ID NO:25, and a light chain variable region comprising thevariable region of amino acid sequence SEQ ID NO: 29; and a bindingmolecule comprising a heavy chain variable region comprising thevariable region of amino acid sequence SEQ ID NO:28, and a light chainvariable region comprising the variable region of amino acid sequenceSEQ ID NO:
 32. 32. The human binding molecule of claim 29, wherein thebinding molecule comprises an immunoconjugate comprising at least onedetectable tag.
 33. A binding molecule capable of binding to andstimulating a human OX40-receptor, produced by a process comprising:culturing under conditions conducive to expression of the bindingmolecule a host comprising at least one vector encoding a bindingmolecule thereof able to bind to and stimulate the human OX40-receptor;expressing the binding molecule; and isolating the binding molecule,wherein the binding molecule is the binding molecule of claim
 29. 34. Anucleic acid encoding the human binding molecule of claim
 29. 35. Avector comprising at least one nucleic acid of claim
 34. 36. A hostcomprising at least one vector of claim
 35. 37. The host of claim 36,wherein the host is a human cell.
 38. A composition comprising: thehuman binding molecule of claim 29, and a stabilizing molecule.
 39. Apharmaceutical composition comprising: the human binding molecule ofclaim 29, and at least one pharmaceutically acceptable excipient. 40.The pharmaceutical composition of claim 35 further comprising: at leastone other therapeutic agent.
 41. A process for producing a bindingmolecule capable of binding and stimulating a human OX40 receptor, themethod comprising: culturing under conditions conducive to theexpression of the binding molecule a host comprising at least one vectorencoding a binding molecule thereof able to bind to and stimulate thehuman OX40-receptor; expressing the binding molecule; and isolating thebinding molecule.
 42. A method for modulating a T cell response in asubject, the method comprising: administering to the subject aneffective dose of a composition comprising the binding molecule of claim29 in an amount sufficient to bind to and stimulate the OX40 receptor inthe subject.
 43. The method according to claim 42, wherein themodulation comprises stimulation of T-cell proliferation.
 44. The methodaccording to claim 42, wherein the subject is a human.